U.S. patent number 11,340,239 [Application Number 16/684,618] was granted by the patent office on 2022-05-24 for bnp (1-32) epitope and antibodies directed against said epitope.
This patent grant is currently assigned to BIO-RAD EUROPE GMBH, CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (C.N.R.S.). The grantee listed for this patent is Bio-Rad Europe GMBH, Centre National De La Recherche Scientifique (C.N.R.S.). Invention is credited to Isabelle Giuliani, Francois Rieunier, Sylvie Villard-Saussine.
United States Patent |
11,340,239 |
Rieunier , et al. |
May 24, 2022 |
BNP (1-32) epitope and antibodies directed against said epitope
Abstract
The present invention relates to a polypeptide carrying a human
BNP(1-32) epitope according to Formula (I):
a.sub.1-R.sub.1-X.sub.1-FGRKMDR-X.sub.2-R.sub.2-a.sub.2 as well as
ligands specific of the FGRKMDR epitope.
Inventors: |
Rieunier; Francois (Bois
d'Arcy, FR), Giuliani; Isabelle (Garches,
FR), Villard-Saussine; Sylvie (Issy les Moulineaux,
FR) |
Applicant: |
Name |
City |
State |
Country |
Type |
Bio-Rad Europe GMBH
Centre National De La Recherche Scientifique (C.N.R.S.) |
Basel
Paris |
N/A
N/A |
CH
FR |
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Assignee: |
BIO-RAD EUROPE GMBH (Basel,
CH)
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (C.N.R.S.)
(Paris, FR)
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Family
ID: |
1000006325323 |
Appl.
No.: |
16/684,618 |
Filed: |
November 15, 2019 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20200072853 A1 |
Mar 5, 2020 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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15288005 |
Oct 7, 2016 |
10495649 |
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12733005 |
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9481720 |
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PCT/EP2008/060188 |
Aug 1, 2008 |
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Foreign Application Priority Data
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Aug 3, 2007 [FR] |
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07 05711 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K
14/58 (20130101); G01N 33/74 (20130101); G01N
2800/50 (20130101); G01N 2800/32 (20130101); G01N
2800/52 (20130101); G01N 2333/58 (20130101); G01N
2800/324 (20130101) |
Current International
Class: |
G01N
33/74 (20060101); C07K 14/58 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2084544 |
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Nov 2006 |
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EP |
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2 135 087 |
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Dec 2009 |
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EP |
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2135087 |
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Dec 2009 |
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EP |
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WO 2004/014952 |
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Feb 2004 |
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WO |
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WO 2006/088700 |
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Aug 2006 |
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WO |
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WO 2007/010256 |
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Jan 2007 |
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WO |
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WO 2008/056034 |
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May 2008 |
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WO |
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WO 2008/125733 |
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Oct 2008 |
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WO |
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Other References
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Precursor Is the Major Immunoreactive Form of BNP in Patients with
Heart Failure" Clinical Chemistry, 2007, pp. 866-873, vol. 53, No.
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applicant .
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immune response to a B and a T cell epitope from the fusion protein
and measles virus" Journal of General Virology, 1992, pp.
1987-1994, vol. 73. cited by applicant .
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histocompatibility complex molecules by a single amino acid residue
contiguous to a derterminant leads to crypticity of the determinant
as well as lack of response to the protein antigen" Proc. Natl.
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applicant .
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129-134, vol. 9. cited by applicant .
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single domains" Nature Biotechnology, Sep. 7, 2005, pp. 1126-1136,
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NT-proBNP)" Aug. 2010, www.hytest.fi, pp. 1-19. cited by
applicant.
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Primary Examiner: Cheu; Changhwa J
Attorney, Agent or Firm: Saliwanchik, Lloyd &
Eisenschenk
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No.
15/288,005, filed Oct. 7, 2016, now U.S. Pat. No. 10,495,649, which
is a continuation of U.S. application Ser. No. 12/733,005, filed
Mar. 26, 2010, now U.S. Pat. No. 9,481,720, which is the national
stage of PCT/EP08/060188 filed Aug. 1, 2008 and published in
English, which has a priority of French no. 07 05711 filed Aug. 3,
2007, hereby incorporated by reference.
Claims
We claim:
1. A method for measuring, in a biological sample, the
concentration of human BNP(1-32) or a derivative of human
proBNP(1-108) containing the sequence FGRKMDR (SEQ ID NO: 8),
comprising: a) contacting the biological sample with a monoclonal
antibody produced by the hybridoma deposited on Apr. 13, 2007 at
the CNCM (Collection Nationale de Cultures de Microorganismes,
Institut Pasteur, 25, rue du Docteur Roux, 75 724 Paris Cedex 15,
France) under registration number CNCM 1-3746 that is specific of
an epitope of the sequence FGRKMDR (SEQ ID NO: 8) under conditions
allowing the formation of antigen-ligand complexes, b) detecting
any complex which may have formed; c) contacting known
concentrations of a multiepitopic calibrator having the following
general formula (III):
t.sub.1-E.sub.1-L.sub.1-E.sub.2[-L.sub.k-1-E.sub.k].sub.n-t.sub.2
(III) wherein: n is an integer between 0 and 8; k is an integer
between 3 and n+2 when n>0; E.sub.1, E.sub.2, and E.sub.k are
different from one another, wherein one of E.sub.1, E.sub.2 and
E.sub.k is a peptide having a sequence of
R.sub.1-X.sub.1-FGRKMDR(SEQ ID NO: 8)-X.sub.2-R.sub.2, wherein
X.sub.1 is absent or present and when present is selected among C
and GC; X.sub.2 is absent or present and when present is selected
among I and IS; R.sub.1 and R.sub.2, which may be the same or
different, present or absent, represent any amino acid or a peptide
chain of 2 to 15 amino acids, provided that said polypeptide of
formula (III) does not include any portion of human BNP(1-32) of
more than 11 amino acids including the sequence GCFGRKMDRIS (SEQ ID
NO: 63), and each of the remaining two from E.sub.1, E.sub.2 and
E.sub.k independently represents a sequence of 3 to 15 amino acids
selected from the sequence of human proBNP(1-108) (SEQ ID NO: 1);
t.sub.1 represents a hydrogen atom, an acetyl group, a peptide
sequence of 1 to 10 amino acids, a peptide sequence of 1 to 10
N-.alpha. acetylated amino acids, a biotinyl or biocytinyl group, a
peptide sequence of 1 to 10 amino acids carrying a biotinyl or
biocytinyl radical, or a linear amino alkyl (C.sub.1-C.sub.10)
carbonyl chain; t.sub.2 represents a hydroxyl radical, an amino
radical, a peptide sequence of 1 to 10 amino acids, a peptide
sequence of 1 to 10 amino acids carrying a terminal amino group, or
a linear or branched amino alkyl (C.sub.1-C.sub.10) carbonyl chain;
and L.sub.1 and L.sub.k-1 which may be the same or different, each
represents a coupling agent that covalently couples the adjacent
peptide chains with said monoclonal antibody; and d) determining
the concentration of human BNP(1-32) or a derivative of human
proBNP(1-108) containing the sequence FGRKMDR (SEQ ID NO: 8) in the
sample.
2. A method for measuring, in a biological sample, the
concentration of human BNP(1-32) or a derivative of human
proBNP(1-108) containing the sequence FGRKMDR (SEQ ID NO: 8),
comprising: a) contacting the biological sample with at least one
monoclonal antibody specific for an epitope of the sequence FGRKMDR
(SEQ ID NO: 8) under conditions allowing the formation of
antigen-antibody complexes, said monoclonal antibody being produced
by the hybridoma deposited on Apr. 13, 2007 at the CNCM (Collection
Nationale de Cultures de Microorganismes, Institut Pasteur, 25, rue
du Docteur Roux, 75 724 Paris Cedex 15, France) under registration
number CNCM I-3746; b) detecting any complex which may have formed;
and c) determining the concentration of human BNP(1-32) or a
derivative of human proBNP(1-108) containing the sequence FGRKMDR
(SEQ ID NO: 8) in the sample.
Description
The Sequence Listing for this application is labeled
"sequences.txt" which was created on Sep. 28, 2016 and is 16 KB.
The entire content of the sequence listing is incorporated herein
by reference in its entirety.
The invention relates to human brain natriuretic peptides (BNP) and
in vitro diagnosis of congestive heart failure in humans. More
particularly, the invention relates to a new epitope present in the
BNP(1-32) molecule, the antibodies directed against said epitope,
in particular the 20G7 monoclonal antibody, a method for the
immunological assay of BNP(1-32) and proBNP(1-108) and respective
fragments thereof using such an antibody, and testing kits for
carrying out said assays.
Congestive heart failure is a common clinical syndrome, in
particular among the elderly. It normally manifests itself in the
form of an insidious triggering of non-specific symptoms, such as
coughing upon physical exertion, fatigue and the appearance of
peripheral oedema. The diagnosis and assessment of the severity of
the affection (graded in stages I to IV NHYA in accordance with the
New York Heart Association) are based on the combined
interpretation of clinical signs and results of specific tests and
examinations (echocardiography, scintigraphy, exercise test,
etc.).
Due to the severity of congestive heart failure and also the high
patient care costs, early diagnosis of this syndrome is obviously
highly desirable as this would contribute to the implementation of
treatments suitable for avoiding or delaying rapid progression of
the syndrome to severe congestive heart failure. It is therefore
necessary to identify those people at risk of congestive heart
failure and/or unfavourable prognosis or subsequent complications.
This would also make it possible to propose the same tools for
(quickly, simply and cost-effectively) therapeutically monitoring
patients undergoing treatment. Nowadays, such methods for the
diagnosis, prognosis and monitoring of congestive heart failure are
in place and are described below, but they have proved to be
somewhat unsatisfactory and are not completely informative.
Acute coronary syndromes (ACS) are also a current major health
problem. They comprise the following heart diseases: Q-wave
myocardial infarction, myocardial infarction with or without
ST-segment elevation, threat of myocardial infarction or unstable
angina.
The diagnosis, prognosis and monitoring of ACSs are also of the
utmost importance in the medical community. The assay of
natriuretic peptides (BNP(1-32), NT-proBNP(1-76), and
proBNP(1-108)) is of high interest in these applications.
The same also applies to cases of dyspnoea (a disease characterised
by breathing difficulties), cerebrovascular accidents (CVA) (also
known as "stroke" or "apoplexy"), and associated pathologies such
as kidney failure and diabetis associated with these
pathologies.
Presymptomatic markers which may predict congestive heart failure
have long been sought after. In this respect, it has been shown
that cardiomyocytes produce and secrete peptides with natriuretic
activity: a peptide of auricular origin, ANP (Atrial Natriuretic
Peptide) discovered in rats by de Bold et al. Life Science 1981,
vol. 28(1): 89-94, and a natriuretic peptide of
auriculo-ventricular origin known as BNP (Brain Natriuretic
Peptide) discovered by the inventors of patent EP 418 308 and by
Sudoh et al. (1988) Nature 332: 78-81 in pigs and in humans.
The precursor of BNP, preproBNP(1-134), is the form of storage of
the molecule inside cardiomyocytes. Said precursor is cleaved
during and/or after secretion thereof in order to release a signal
peptide and proBNP(1-108). ProBNP(1-108) consists in a polypeptide
of 108 amino acids of the sequence:
TABLE-US-00001 (SEQ ID NO: 1)
H.sub.1PLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTG
VWKSREVATEGIRGHRKMVLYTLRAPR.sub.76S.sub.77PKMVQGSGCFGRKMDRISS
SSGLGCKVLRRH.sub.108.
It is cleaved before and/or during secretion thereof, between the
Arg.sub.76 and Ser.sub.77 amino acids into, on the one hand, BNP,
also known as BNP(77-108) or BNP-32, or even BNP(1-32) (the term
which will be used hereinafter), and the N-terminal part of the
prohormone, BNP(1-76), also known as the N-terminal fragment of
proBNP or NT-proBNP(1-76) (term which will be used
hereinafter).
BNP(1-32), the vasoactive form of the molecule, consists in a
peptide of 32 amino acids of the sequence:
TABLE-US-00002 (SEQ ID NO: 2)
S1PKMVQGSGCFGRKMDRISSSSGLGCKVLRRH32.
17 amino acids form a loop closed by a disulphide bond between the
two oxidised cysteine residues (C.sub.10 and C.sub.26), said loop
being surrounded upstream by 9 amino acids (which constitute the
N-terminal part), and downstream by 6 amino acids (which constitute
the C-terminal part).
The integrity of the loop is important for obtaining good
biological activity. Of the 17 residues forming the loop, 11 are
also conserved in the 2 other natriuretic peptides, which are ANP
(A-type natriuretic peptide) and CNP (C-type natriuretic
peptide).
NT-proBNP(1-76) is formed by the 76 N-terminal amino acids of
proBNP(1-108) and has the following sequence:
TABLE-US-00003 (SEQ ID NO: 3)
H.sub.1PLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTG
VWKSREVATEGIRGHRKMVLYTLRAPR.sub.76.
Interestingly, these 3 polypeptides, proBNP(1-108), NT-proBNP(1-76)
and BNP(1-32), have proved to be good markers of congestive heart
failure in such a way that different assays with specific
combinations of antibodies have been developed.
Indeed, as proBNP(1-108) has been recognised as circulating in the
blood since the initial studies of Tateyama et al. (1992)
Biochemical and Biophysical Research Communications 185: 760-767,
different immunological assays for detecting proBNP(1-108) have
been suggested. Patent application WO 99/13331 thus describes a
sandwich assay of proBNP(1-108) with the aid of an antibody which
recognises the 1-76 portion of proBNP(1-108) and an anti-BNP(1-32)
antibody. This type of assay lacks sensitivity due to the binding,
onto the solid phase (capture antibodies), of by-products of
cleaved proBNP(1-108) having the epitope recognised by the capture
antibody, that is to say, NT-proBNP(1-76) and the products
resulting from the progressive cleavage thereof.
Application WO 2004/14952 describes the detection of proBNP(1-108)
with the aid, on the one hand, of an antibody which recognises an
epitope of the sequence RAPRSP, located at the hinge of
NT-proBNP(1-76) and BNP(1-32) (also called hinge 76 antibody) and,
on the other hand, an anti-BNP(1-32) antibody in such a way that,
in this type of assay, the assay is specific to the prohormone
only, i.e. there is no cross-reactivity with the two forms of
NT-proBNP(1-76) and BNP(1-32).
With regard to the assay of NT-proBNP(1-76), application WO
93/24531 describes a method for in vitro diagnosis of congestive
heart failure based on the detection of NT-proBNP(1-76). However,
the method described in application WO 93/24531 does not appear to
be easily carried out on NT-proBNP(1-76) in blood samples. Indeed,
the only examples shown were carried out, not on human sera, but on
standard ranges obtained using a synthetic peptide, the peptide
NT-proBNP(47-64). In order to overcome this drawback, a highly
sophisticated automated system has since proven to be
necessary.
Finally, a plurality of assays of BNP(1-32) have been developed
using various antibodies. For example, patent JP 2 676 114 B2
describes 2 monoclonal antibodies (KY-hBNPI and KY-hBNPII) which
recognise the cyclic structure of BNP(1-32), with no other
details.
In addition, patent application EP 542 255 describes a monoclonal
antibody which recognises the histidine H.sub.32, last amino acid
of the C-terminal K.sub.27VLRRH.sub.32 epitope of BNP(1-32). Other
epitopes present on BNP(1-32) are known: 3 epitopes of the sequence
.sub.1SPKMVQGSGC.sub.10 (SEQ ID NO: 22), .sub.5VQGSGCFGR.sub.13
(SEQ ID NO: 21), and .sub.15MDRISSSSGLG.sub.25 (SEQ ID NO: 23) are
also described as being highly immunogenic in application WO
97/32900 and patent U.S. Pat. No. 6,162,902. Said documents also
describe monospecific antibodies directed against these
epitopes.
In 2005, the HyTest company (Turku, Finland) put various antibodies
specific of BNP(1-32) on the market. As described in the article by
Seferian et al. (2007) Clinical Chemistry 53:5, 866-873, some of
these anti-BNP(1-32) antibodies target the 1-10, 11-22, 17-23 or
26-32 region of BNP(1-32). Among the 17 antibodies obtained by
immunising Balb/c mice using the .sub.11FGRKMDRISSSS.sub.22 (SEQ ID
NO: 61) peptide of BNP(1-32), only the 24C5 and the 26E2 monoclonal
antibodies are described. However, their epitopes are not precisely
characterised: it is only indicated that they are directed against
the above mentioned sequence of amino acids 11 to 22 of
BNP(1-32).
Patent application WO 2006/88700 describes another epitope present
on BNP(1-32). It has the sequence of amino acids
R.sub.13(K.sub.14)(M.sub.15)D.sub.16R.sub.17I.sub.18 (SEQ ID NO:
24), included in the amino acids 13-20 which form part of the
cyclic structure of human BNP(1-32). This application also
describes a monoclonal antibody, designated 3-631-436, which
recognises this epitope specifically. The 4 amino acids R.sub.13,
D.sub.16, R.sub.17, and I.sub.18 are described as being
functionally significant for binding the 3-631-436 antibody to this
epitope. The amino acids located upstream this epitope (such as
phenylalanine F.sub.11 and glycine G.sub.12) are not mentioned.
One of the drawbacks of the BNP(1-32) hormone is that it is
unstable in plasma and in serum. Indeed, protease-type enzymes seem
to cleave BNP(1-32). For example, Shimizu et al. (2002) Clinica
Chimica Acta 316: 129-135 report that the N-terminal part of
BNP(1-32), more particularly the Pro.sub.2-Lys.sub.3 bond, would be
cleaved by proteases, as well as the Arg.sub.30-Arg.sub.31 bond in
the C-terminal position. Boerrigter et al. (2007) Am. J. Physiol.
Regul. Integr. Comp. Physiol. 292: R897-90 and Hawkridge et al.
(2005) Proc. Natl. Acad. Sci. USA. 102:17442-7 have described, in
particular, the degradation of BNP(1-32) in N-terminal
position.
Likewise, some bond cleavages caused by endopeptidases have been
reported (Davidson & Struthers (1994) J. Hypertension 13:
329-336). Therefore, the assay of BNP(1-32) calls for specific
precautions (Davidson et al. (1995) Circulation 91:1276-7;
Gobinet-Georges et al. (2000) Clin. Chem. Lab. Med. 38:519-23), and
implies a suitable choice of antibodies.
Recently, several teams have shown that natriuretic peptides could
circulate in glycosylated and/or truncated form (Schellenberger
(2006) Arch. Biochem. Biophys. 451: 160-6; Liang et al. (2007)
Journal of the American College of Cardiology 49: 1071-8; Lam et
al. (2007) Journal of the American College of Cardiology 49:
1193-292).
Wth regard to early diagnosis of congestive heart failure, ACSs,
dyspnoea and other cardiovascular diseases as well as CVAs and
associated pathologies, such as diabetis and kidney failure, there
is always a need to improve the reagents and methods for detecting
BNP(1-32) and proBNP(1-108), in particular taking into account the
problem of the instability of BN P(1-32).
DESCRIPTION OF THE INVENTION
The present invention follows mainly from the entirely unexpected
finding by the inventors of an unknown and unsuspected epitope
which exists in the human BNP(1-32) molecule and a specific
monoclonal antibody which recognises said epitope. Contrary to all
expectations, they discovered that the sequence
F.sub.11GRKMDR.sub.17 (SEQ ID NO: 51) of BNP(1-32) constitutes a
beneficial epitope for generating antibodies which recognise the
cyclic structure (amino acids 10-26) of BNP(1-32) and they obtained
a monoclonal antibody, called 20G7, which specifically recognises
said F.sub.11G.sub.12RK.sub.14MDR.sub.17 (SEQ ID NO: 51) epitope.
In other words, the 20G7 antibody is a monoclonal antibody specific
of the F.sub.11GRKMDR.sub.17 (SEQ ID NO: 51) epitope of
BNP(1-32).
The present invention also provides an immunoassay method for
detecting BNP(1-32) and proBNP(1-108) as well as circulating
fragments thereof using the 20G7 monoclonal antibody and reagents
containing said antibody.
Indeed, the inventors noticed that, while synthesizing peptides
located in the cyclic region (amino acids 10-26) of human BNP(1-32)
and immunising mice with said peptides, some resulting antibodies
only reacted with peptides of this type if said peptides contained
phenylalanine F.sub.11 residues, lysine K.sub.14 and Arginine
R.sub.17 and that isoleucine I.sub.18, which was particularly
significant in the epitope described in the international
application WO2006/88700, did not belong to the epitope according
to the present invention.
For example, the inventors immunised some mice with the
TGCFGRKMDRISTSTAIGCKVL (SEQ ID NO: 4) peptide and others with the
SGCYGRKMDRISTSTAIGCKVL (SEQ ID NO: 5) peptide. They observed that
the immune response to BN P(1-32) was much greater in the mice
immunised with the TGCFGRKMDRISTSTAIGCKVL (SEQ ID NO: 4) peptide
than in the mice immunised with the SGCYGRKMDRISTSTAIGCKVL (SEQ ID
NO: 5) peptide. It should be noted that, in both cases, cysteines
are presented in oxidised form via an intrachain disulphide
bond.
After lymphocytic fusion of immunised mice spleen cells with
myeloma cells, the inventors were thus able to produce different
hybrid clones. In particular, they obtained a monoclonal antibody,
called 20G7-15 Mar. 2007 (hereinafter, referred to as "20G7" for
convenience), which only recognises those peptides of BNP(1-32) and
proBNP(1-108) which contain the residues phenylalanine F.sub.11,
lysine K.sub.14 and arginine R.sub.17. In fact, these amino acids
F.sub.11, K.sub.14 and R.sub.17 have proven to be important for
optimal binding of the 20G7 monoclonal antibody to BNP(1-32) and to
proBNP(1-108) as well as to respective fragments thereof.
The hybridoma which secretes the 20G7-15 Mar. 2007 (20G7)
monoclonal antibody was deposited on Apr. 13, 2007 by Bio-Rad at
the CNCM (Collection Nationale de Cultures de Microorganismes,
Institut Pasteur, 25, rue du Docteur Roux, 75 724 Paris Cedex 15,
France) under registration number CNCM I-3746.
Surprisingly and unexpectedly, the inventors observed with the 20G7
antibody that, as soon as the residues F.sub.11, K.sub.14 and
R.sub.17 were substituted by an alanine, either individually or
jointly, the antigenic reactivity of the peptide, compared to that
of the natural SGCFGRKMDRISSSSGLGCKVL (SEQ ID NO: 6) peptide, was
considerably affected, whereas substituting other amino acids of
the epitope had almost no effect on the antigenic reactivity of the
peptide. Also, an in-depth study on the 20G7 antibody has shown
that it recognises the F.sub.11GRKMDR.sub.17 (SEQ ID NO: 51)
epitope, but does not recognise the A.sub.11GRKMDR.sub.17 (SEQ ID
NO: 62) sequence nor the GRKMDR.sub.17O.sub.18 (SEQ ID NO: 52)
sequence, nor the C.sub.10F.sub.11GRKMD (SEQ ID NO: 50)
sequence.
The 20G7 monoclonal antibody thus recognises the
F.sub.11GRKMDR.sub.17 (SEQ ID NO: 51) epitopic sequence of
BNP(1-32), but does not substantially recognise the above mentioned
VQGSGCFGR (SEQ ID NO: 21), SPKMVQGSGC (SEQ ID NO: 22), and
MDRISSSSGLG (SEQ ID NO: 23)) epitopes of application WO 97/32900,
nor the R(K)(M)DRI (SEQ ID NO: 24) epitope of application
WO2006/88700.
The present invention therefore relates to a polypeptide carrying a
human BNP(1-32) epitope having the formula (I):
a.sub.1-R.sub.1-X.sub.1-FGRKMDR-X.sub.2-R.sub.2-a.sub.2 (SEQ ID NO:
51) (I) wherein a.sub.1 may be H or represent a functional group or
chemical group selected from a thiol, alcohol, aminoxy, primary
amine or secondary amine functional group, an amino carboxyl group,
a biotinyl group and an acetyl group; a.sub.2 may represent an OH,
NH.sub.2 functional group or an alcoxyl group (as will be clear for
the man skilled in the art a.sub.2 is attached to the carbonyl
(--CO--) moiety of the acidic function of the last amino acid of
the polypeptide); X.sub.1 is absent or present and when present is
selected among C and GC; X.sub.2 is absent or present and when
present is selected among I and IS; R.sub.1 and R.sub.2, which may
be the same or different, absent or present, represent any amino
acid or a peptide chain of 2 to 15 amino acids, provided that said
polypeptide of formula (I) does not include any portion of human
BNP(1-32) of more than 11 amino acids including the sequence
GCFGRKMDRIS (SEQ ID NO: 63).
As an equivalent alternative, formula (I) can also be defined as
follows:
a.sub.1-(R.sub.1)-(G)-(C)-FGRKMDR-(I)-(S)-(R.sub.2)-a.sub.2(SEQ ID
NO: 51) wherein a.sub.1, a.sub.2, R.sub.1 and R.sub.2 are as
defined above.
"Epitope" or "epitopic site" means an amino acid sequence which is
recognised by at least one antibody and allows the antibody to bind
specifically to said amino acid sequence.
In a preferred embodiment, R.sub.1 and R.sub.2 may be coupled to
carrier molecules, reagents or marker molecules.
In another preferred embodiment, said polypeptide is selected in
the group consisting of a.sub.1-SGCFGRKMDR-a.sub.2 (SEQ ID NO: 33),
a.sub.1-GCFGRKMDRI-a.sub.2 (SEQ ID NO: 34),
a.sub.1-CFGRKMDRIS-a.sub.2 (SEQ ID NO: 35) and
a.sub.1-FGRKMDRISS-a.sub.2 (SEQ ID NO: 36), where a.sub.1 and
a.sub.2 are as defined above.
In another preferred embodiment, the polypeptide as defined above
corresponds to formula (II): a.sub.1-FGRKMDR-a.sub.2(SEQ ID NO: 51)
(II) wherein a.sub.1 and a.sub.2 are as defined above.
The present invention also relates to the use of a polypeptide as
defined above, for the preparation of ligands directed against
human BNP(1-32) or human proBNP(1-108) as well as the respective
fragments thereof comprising the sequence FGRKMDR (SEQ ID NO:
51).
"Fragment of proBNP(1-108)" according to the invention means any
fragment which is smaller than proBNP(1-108), including, in
particular, BNP(1-32). For example, the proBNP(3-108) fragment, as
described in Lam et al. (2007) J. Am. Coll. Cardiol. 49:1193-1202
and produced by cleavage by a dipeptidase. The term "fragment of
proBNP(1-108)" according to the invention also includes any
polypeptide having been subjected to at least one
post-translational modification of proBNP(1-108), such as
phosphorylation, glycosylation or the like. For example,
Schellenberger et al. (2006) Arch. Biochem. Biophys. 51:160-6 have
shown that proBNP(1-108) is a glycoprotein which is O-glycosylated
either entirely or in part.
"Fragment of BNP(1-32)" according to the invention means any
fragment which is smaller than BNP(1-32). In this case also,
degradation of BNP(1-32) has already been reported in the
literature: for example, the BNP(3-32) fragment has been described
by Lam et al. (supra) and by Hawkridge et al. (2005) Proc. Natl.
Acad. Sci. USA 102:17442-7.
The terms "proBNP(1-108)" and "BNP(1-32)" as well as "fragment of
proBNP(1-108)" and "fragment of BNP(1-32)" also include any
polypeptide having been subjected to at least one
post-translational modification, such as phosphorylation,
glycosylation or the like. For example, Schellenberger et al.
(2006) Arch. Biochem. Biophys. 51:160-6 have shown that
proBNP(1-108) is a glycoprotein which is O-glycosylated either
entirely or in part.
"Ligands directed against human BNP(1-32) or human proBNP(1-108) as
well as respective fragments thereof comprising the sequence
FGRKMDR" (SEQ ID NO: 51) refers to any molecule able to bind
specifically to human BNP(1-32), to human proBNP(1-108) or to
fragments thereof.
The term "specific", when it refers to recognition of a ligand or
binding of a ligand to a target, means that the ligand interacts
with the target without interacting substantially with another
target which does not structurally resemble the target. "Specific"
recognition of the FGRKMDR (SEQ ID NO: 51) epitope means that the
interaction of the ligand with a target comprising the epitope does
not substantially involve antigenic determinants, in particular
amino acids, other than those of the epitope. In particular, this
means that the ligand is able to bind a sequence of amino acids of
the BNP(1-32) and/or proBNP(1-108) sequence as well as respective
fragments thereof comprising the amino acids comprising the FGRKMDR
(SEQ ID NO: 51) epitope, but is unable to bind a sequence of amino
acids of the BNP(1-32) and/or proBNP(1-108) sequence which does not
comprise the FGRKMDR (SEQ ID NO: 51) epitope in its entirety.
In addition, an amino acid present in an epitope is said to be
"critical" as soon as its substitution by an alanine leads to a
reduction of at least 50% in the antigenicity of said epitope,
according to Laune et al. (2002) Journal of Immunological Methods
267:53-70.
Moreover, an amino acid present in an epitope is said to be
"essential" as soon as its substitution by an alanine leads to a
reduction of at least 80% in the antigenicity of said epitope.
Preferably, a ligand which specifically recognises the FGRKMDR (SEQ
ID NO: 51) epitope according to the invention does not interact
substantially with peptides having the sequence VQGSGCFGR (SEQ ID
NO: 21), SPKMVQGSGC (SEQ ID NO: 22), MDRISSSSGLG (SEQ ID NO: 23),
RKMDRI (SEQ ID NO: 24) and RKMDRISS (SEQ ID NO: 25).
The expression "does not interact substantially with peptides
having the sequence VQGSGCFGR (SEQ ID NO: 21), SPKMVQGSGC (SEQ ID
NO: 22), MDRISSSSGLG (SEQ ID NO: 23), RKMDRI (SEQ ID NO: 24) and
RKMDRISS (SEQ ID NO: 25)" means that the ligand has a cross
reaction with one or other of these sequences of less than 20%,
preferably less than 10%, more preferably less than 5%,
particularly preferably less than 2%.
Within the scope of specific recognition of a target, binding
constants greater than 10.sup.6 M.sup.-1 are preferred, binding
constants greater than 10.sup.8 M.sup.-1 are more preferred and
binding constants greater than 10.sup.10 M.sup.-1 are particularly
preferred.
Preferably, the residues F.sub.11, K.sub.14 and R.sub.17 are also
essential for binding a ligand according to the invention to the
epitope, since their substitution by an alanine is characterised by
a loss of 82%, 95% and 85% respectively in the binding of the
monoclonal antibody produced by the hybridoma deposited on April
13, 2007 by Bio-Rad at the CNCM (Collection Nationale de Cultures
de Microorganismes, Institut Pasteur, 25, rue du Docteur Roux, 75
724 Paris Cedex 15, France) under registration number CNCM I-3746,
to the epitopic peptide.
Preferably, the ligand is selected from the group constituted by an
antibody or a fragment of said antibody which recognises the
epitope, an aptamer, and a polypeptide which specifically
recognises the epitope obtainable by phage display.
In this context, the term "antibody" refers to any polyclonal or
monoclonal antibody.
The fragments scFv, Fab, Fab', F(ab').sub.2, as well as camelids
single-chain antibodies are examples of antibody fragments which
recognise the epitope.
The "aptamers" are well-known by the one skilled in the art.
Aptamers are compounds of a nucleotide, in particular a
ribonucleotide or desoxyribonucleotide, or a peptide nature able to
bind specifically to a target, in particular a protein target. The
aptamers of a nucleotide nature and the production thereof are
described, in particular, by Ellington et al. (1990) Nature
346:818-22 and Bock et al. (1992) Nature 355:564-6. The aptamers of
a peptide nature and the production thereof are described, in
particular, by Hoppe-Seyler et al. (2000) J. Mol Med.
78:426-30.
"Phage display" denotes a technique for selecting polypeptide
ligands expressed on the capsid of a bacteriophage and encoded by a
nucleic sequence inserted into the capsid encoding gene. This
method is well known by the one skilled in the art and is
described, in particular, by Scott & Smith (1990) Science
249:386-390, and Marks et al. (1991) J. Mol. Biol. 222:581-597.
Preferably, the polypeptide obtainable by phage display is an
scFv-type polypeptide (single-chain variable fragment). This
technique is described, in particular, by Winter et al. (1994)
Annu. Rev. lmmunol. 12:433-455.
The ligands may also be obtained by chemical synthesis or by
genetic engineering.
Preferably, the polypeptides as defined above are used to prepare
antibodies, in particular monoclonal antibodies.
In this context, the invention also relates to use of a polypeptide
as defined above for the preparation of a hybridoma which secretes
a monoclonal antibody directed against human BNP(1-32) or human
proBNP(1-108) as well as the respective fragments thereof
comprising the sequence FGRKMDR (SEQ ID NO: 51).
The invention thus also relates to a method for preparing a
hybridoma which secretes a monoclonal antibody directed against
human BNP(1-32) or human proBNP(1-108) as well as the respective
fragments thereof comprising the sequence FGRKMDR (SEQ ID NO:
51),
wherein:
samples of lymphocytes secreting immunoglobulins are taken from an
animal, such as a mouse, rabbit or rat, immunised with a
polypeptide as defined above, the lymphocytes are then fused with
myeloma cells, such as Sp2 myeloma cells (ATCC CRL-1581), in order
to obtain a hybridoma.
The present invention also relates to a hybridoma obtainable by the
method for preparing a hydridoma defined above.
More particularly, the present invention relates to the hybridoma
deposited on Apr. 13, 2007 at the CNCM (Collection Nationale de
Cultures de Microorganismes, Institut Pasteur, 25, rue du Docteur
Roux, 75 724 Paris Cedex 15, France) under registration number CNCM
I-3746.
Generally, the methodology used to obtain hybridomas and monoclonal
antibodies may follow the conventional method of lymphocyte fusion
and hybridoma culture described by Kohler & Milstein (1975)
Nature 256:495-497. Other methods for preparing monoclonal
antibodies are also known (e.g., Harlow et al., ed. 1988
"Antibodies: a laboratory manual").
Alternative methods to this conventional method also exist.
Monoclonal antibodies can be produced, for example, by expressing a
nucleic acid cloned from a hybridoma.
The present invention also relates to a ligand specific of an
epitope of the sequence FGRKMDR (SEQ ID NO: 51).
Preferably, the ligand is selected from the group constituted by an
antibody or a fragment of said antibody which recognises the
epitope, an aptamer, and a polypeptide which specifically
recognises the epitope obtained by phage display.
More preferably, the ligand is constituted by an antibody which
specifically recognises an epitope of the sequence FGRKMDR (SEQ ID
NO: 51), or a fragment of said antibody which specifically
recognises the epitope. Even more preferably, the ligand is
constituted by a monoclonal antibody, in particular a monoclonal
antibody produced by a hybridoma as defined above.
The invention thus relates, in particular, to a ligand as defined
above, constituted by the monoclonal antibody produced by the
hybridoma deposited on Apr. 13, 2007 at the CNCM (Collection
Nationale de Cultures de Microorganismes, Institut Pasteur, 25, rue
du Docteur Roux, 75 724 Paris Cedex 15, France) under registration
number CNCM I-3746.
The invention further more particularly relates to a ligand as
defined above harbouring at least one Complementary Determining
Region (CDR), in particular all the CDR, of the above-defined
ligand constituted by the monoclonal antibody produced by the
hybridoma deposited on Apr. 13, 2007 at the CNCM (Collection
Nationale de Cultures de Microorganismes, Institut Pasteur, 25, rue
du Docteur Roux, 75 724 Paris Cedex 15, France) under registration
number CNCM I-3746. CDR and methods for transferring a CDR from an
antibody to a ligand, preferably another antibody, are well known
to the man skilled in the art and are notably described e.g. in
Nicaise et al. (2004) Protein Science 13:1882-1891 or Kettleborough
et al. (1991) Protein Engineering 4:773-783.
In addition, the ligands as defined above may be coupled to carrier
molecules, reagents or labelled molecules.
The present invention also relates to a ligand as defined above for
detecting, in a biological sample, human BNP(1-32) or human
proBNP(1-108) as well as the respective fragments thereof
comprising the sequence FGRKMDR (SEQ ID NO: 51).
In fact, the present invention also relates to a method for
detecting, in a biological sample, human BNP(1-32) or human
proBNP(1-108) as well as respective fragments thereof containing
the sequence FGRKMDR (SEQ ID NO: 51), comprising: 1) contacting the
biological sample with at least one ligand as defined above,
preferably under conditions allowing the formation of
antigen-ligand complexes, and 2) detecting any complexes which may
have formed.
In the context of the invention, a "biological sample" or even a
"biological fluid sample" is preferably constituted by a biological
liquid, such as blood, plasma, serum, cerebrospinal fluid, saliva,
urina and lacrima, etc. (see e.g. Michielsen et al., (2008) Ann
Clin Biochem 45:389-94; Cortes et al., (2006) Eur J Heart Fail
8:621-7; Kirchhoff et al., (2006) J Neurotrauma 23:943-9; Kaneko et
al., (1993) Brain Res 612:104-9). As it is meant in this case, the
term "biological sample" includes both the sample as taken and the
sample which has been subjected to various treatments, in
particular to render it suitable for use in the processes and
methods according to the invention.
In a preferred embodiment, the above detection method comprises at
least one additional step of contacting the biological sample with
at least one additional ligand specific of human BNP(1-32) or human
proBNP(1-108) and of the respective fragments thereof, with has a
different specificity from that of the ligand according to the
invention.
Preferably, the additional ligand is an antibody.
The present invention also relates to a method of diagnosis,
prognosis, risk stratification or therapeutic follow-up of at least
one cardiac and/or vascular pathology in an individual, comprising
the following steps of: 1) contacting a biological sample from the
individual with at least one ligand as defined above, preferably
under conditions allowing the formation of antigen-ligand
complexes, 2) detecting any complex which may have formed, and, 3)
based on the result of the detection in step 2, determining a
diagnosis, a prognosis, a risk of the development or therapeutic
follow-up of the pathology in the individual.
In a particular embodiment, the method defined above comprises at
least one additional step of contacting the biological sample with
at least one additional ligand specific of human BNP(1-32), or a
human proBNP(1-108) derivative, which has a different specificity
to that of the ligand according to the invention.
Preferably, the additional ligand is an antibody.
Preferably, the pathology is selected from the group constituted
of: congestive heart failure, acute coronary syndrome,
cerebrovascular accident, kidney failure, dyspnea, high blood
pressure, atheromatous plaque rupture, patent ductus arteriosus in
premature newborns, and/or diabetis.
"Congestive heart failure" means the pathological state in which an
anomaly of the cardiac function is responsible for the heart being
unable to pump blood sufficiently to satisfy the metabolic needs of
the organism and/or in which the heart fulfils needs but with
abnormally high filling pressures. In particular, it may relate to
a left and/or right ventricular failure.
"Acute Coronary Syndromes" denotes two categories in particular:
acute coronary insufficiency accompanied by a persistent upslope
[i.e. elevation] of the ST segment revealing the formation of a
Q-wave transmural infarction corresponding generally to an acute
total coronary occlusion, and acute coronary insufficiency with no
upslope [i.e. with no elevation] of the ST segment corresponding to
non-Q-wave infarction, also known as unstable anginas which
correspond to plaque ruptures and incomplete thromboses and require
different treatment.
"Dyspnea" means the pathological state characterised by breathing
difficulties accompanied by feelings of obstruction or tightness.
It is an extremely common symptom which may be due to several
causes. Only a methodical approach enables appropriate
treatment.
According to the definition of the World Health Organisation,
"cerebrovascular accident" or "CVA" or "stroke" or "apoplexy" means
the pathological state characterised by the rapid development of
localised or global clinical signs of cerebral dysfunction
accompanied with symptoms lasting more than 24 hours, which may
result in death, with no apparent cause other than a vascular
origin.
The process and the method above may be carried out in accordance
with various formats well-known to the one skilled in the art, for
example in solid or homogeneous phase, in one or two steps, by a
sandwich method or by a competitive method.
Preferably, the sandwich method in solid phase between 2 ligands
(preferably antibodies), one being a capture ligand and the other
being a detection ligand, will be used. This type of immunoassay is
particularly well-known to the one skilled in the art. For example,
the article by Seferian et al. (2007) Clin. Chem. 53:866-873 gives
an example of a sandwich immunoassay (or immunometric assay at 2
sites) for assaying BNP(1-32) and proBNP(1-108), each time using a
pair of antibodies (an antibody immobilised in solid phase and an
labelled antibody in detection).
"Capture ligand" means a ligand capable of binding the BNP(1-32)
and/or proBNP(1-108) antigen, as well as the respective fragments
thereof, present in the biological sample.
The presence of the antigen in the biological sample is revealed by
detection means, in particular a "detection ligand". A detection
ligand, which is labelled, is able to bind to the captured antigen,
by recognising an epitopic site which is different from that
recognised by the capture ligand.
The term "labelled" refers both to a direct labelling and to an
indirect labelling (for example, by means of other ligands,
themselves directly labelled, or using reagents of a labelled
"affinity pair", such as, but not exclusively, the labelled
avidin-biotin pair, etc.).
In the case of the sandwich method, the capture ligand is
preferably selected in such a way that it specifically recognises
an epitope on the natural antigen of the patient, whilst the
detection ligand is selected preferably in such a way that it
specifically recognises another epitope on the natural antigen of
the patient.
Preferably, the capture ligand is immobilised on a solid phase. By
way of non-limiting examples of solid phase, microplates could be
used, in particular polystyrene microplates, such as those sold by
Nunc, Denmark. Solid particles or beads, paramagnetic beads, such
as those produced by Dynal, Merck-Eurolab (France) (under the
trademark Estapor.TM.) and Polymer Laboratories, or even
polystyrene or polypropylene test tubes, glass, plastic or silicon
chips, etc. may also be used.
ELISA assays, radioimmunoassays, or any other detection method may
be used to reveal the presence of formed antigen-antibody
complexes. Thus, different types of labelling of ligands in
particular of antibodies, are possible (radioactive, ezymatic,
fluorescent, etc.).
The detection may also be carried out by new methods based on mass
accumulation, such as surface plasmon resonance (SPR), by
piezo-electric detection, but also by mass spectrometry or any
other methods defined as enabling the study of a
ligand-antigen-type interaction in the absence of a second labelled
ligand.
A preferred implementation of the above process or method consists
in using a ligand as defined above immobilised on a solid phase in
combination with at least one monoclonal or polyclonal antibody
directed against the N-terminal portion of BNP(1-32), present in a
labelled form.
Another preferred implementation of the above process or method
consists in using a ligand as defined above immobilised on a solid
phase in combination with at least one monoclonal or polyclonal
antibody directed against the C-terminal portion of BNP(1-32), such
as the 50B7 antibody, (specific of the 26-32 peptide of BNP(1-32)),
available from HyTest, present in a labelled form.
Alternatively, according to yet another preferred implementation of
the above method or process, a ligand as defined above may be used,
in a labelled form, in combination with at least one monoclonal or
polyclonal antibody directed against the N-terminal or C-terminal
portion of BN P(1-32), present in an immobilised form on a solid
phase.
A preferred implementation of the above method or process consists
in using a ligand as defined above immobilised on a solid phase in
combination with at least one monoclonal or polyclonal antibody
directed against the N-terminal portion of NT-proBNP(1-108), such
as the 16F3 antibody, (specific of the 13-20 peptide of NT-proBNP),
available from HyTest, present a labelled form.
Alternatively, a preferred implementation of the above method or
process consists in using a ligand as defined above a labelled form
in combination with at least one monoclonal or polyclonal antibody
directed against the N-terminal portion of proBNP(1-108), such as
the 16F3 antibody, (specific of the 13-20 peptide of NT-proBNP),
available from HyTest, present in an immobilised form on a solid
phase.
Another preferred implementation of the above method or process
consists in using a ligand as defined above immobilised on a solid
phase in combination with a monoclonal antibody directed against
the RAPR.sub.76S.sub.77P(SEQ ID NO: 55) sequence of proBNP(1-108)
(such as the one described in patent application WO2004/14952, or
in Giuliani et al. (2006) Clin. Chem.52:1054-1061), present in a
labelled form.
Alternatively, a preferred implementation of the above method or
process consists in using a ligand as defined above in a labelled
form in combination with a monoclonal antibody directed against the
RAPR.sub.76S.sub.77P(SEQ ID NO: 55) sequence of proBNP(1-108) (such
as the one described in patent application WO2004/14952, or in
Giuliani et al. (2006) Clin. Chem.52:1054-1061), present in an
immobilised form on a solid phase.
The present invention also relates to a multiepitopic calibrator
having the following general formula (III):
t.sub.1-E.sub.1-L.sub.1-E.sub.2[-L.sub.k-1-E.sub.k].sub.n-t.sub.2
(III) wherein: n is an integer between 0 and 8; k is an integer
between 3 and n+2 when n>0; E.sub.1, E.sub.2, and E.sub.k are
different from one another, one representing a
R.sub.1-X.sub.1-FGRKMDR-X.sub.2-R.sub.2 (SEQ ID NO: 51) peptide
sequence, wherein X.sub.1, X.sub.2, R.sub.1 and R.sub.2 are as
defined above, and the others representing a sequence of 3 to 15
amino acids selected from the sequence of human proBNP(1-108);
t.sub.1 represents a hydrogen atom, an acetyl group, a peptide
sequence of 1 to 10 amino acids, a peptide sequence of 1 to 10
N-.alpha. acetylated amino acids, a biotinyl or biocytinyl group, a
peptide sequence of 1 to 10 amino acids carrying a biotinyl or
biocytinyl radical, or a linear amino alkyl (C.sub.1-C.sub.10)
carbonyl chain; t.sub.2 represents a hydroxyl radical, an amino
radical, a peptide sequence of 1 to 10 amino acids, a peptide
sequence of 1 to 10 amino acids carrying a terminal amino group, or
a linear or branched amino alkyl (C.sub.1-C.sub.10) carbonyl chain
(as will be clear for the man skilled in the art t.sub.2 is
attached to the carbonyl (--CO--) moiety of the acidic function of
the last amino acid of the E.sub.n peptide chain); L.sub.1 and
L.sub.k, which may be the same or different, represent a binding
group of peptide chains.
Preferably, the above multiepitopic calibrator corresponds to the
following general formula (IV):
t.sub.1-E.sub.1-L.sub.1-E.sub.2-L.sub.2-E.sub.3-t.sub.2 (IV)
wherein E.sub.1, E.sub.2, E.sub.3, L.sub.2, t.sub.1 and t.sub.2 are
as defined above.
Preferably, the above multiepitopic calibrator corresponds to the
following general formula (V):
t.sub.1-E.sub.1-L.sub.1-E.sub.2-t.sub.2 (V) wherein E.sub.1,
E.sub.2, L.sub.1, t.sub.1, and t.sub.2, are as defined above.
The above standards (or calibrators) are used to establish standard
curves for the assays of BNP(1-32), proBNP(1-108) and/or one of the
aforementioned fragments thereof. One advantage of said calibrators
is, in particular, their stability.
Preferably, when used for assaying BNP(1-32), a biepitopic standard
according to the invention comprises the FGRKMDR (SEQ ID NO: 51)
epitope according to the invention and another, different epitope
which is selected from the sequence of amino acids 77-108 of
proBNP(1-108).
Preferably, when used for assaying proBNP(1-108), a biepitopic
calibrator according to the invention comprises the FGRKMDR (SEQ ID
NO: 51) epitope according to the invention and another, different
epitope which is selected from the sequence of amino acids 1-76 of
proBNP(1-108) so as to ensure the specificity of the
proBNP(1-108).
Preferably, when used for assaying BNP(1-32), a triepitopic
calibrator according to the invention comprises the
R.sub.1-X.sub.1-FGRKMDR-X.sub.2-R.sub.2 (SEQ ID NO: 51) epitope
according to the invention and two other, different epitopes which
are selected from the sequence of amino acids 77-108 of
proBNP(1-108).
Preferably, when used for assaying proBNP(1-108), a triepitopic
calibrator according to the invention comprises the
R.sub.1-X.sub.1-FGRKMDR-X.sub.2-R.sub.2 (SEQ ID NO: 51) epitope
according to the invention and two other, different epitopes which
are selected from the sequence of amino acids of 1-108
proBNP(1-108).
Preferably, in the above calibrator,
R.sub.1-X.sub.1-FGRKMDR-X.sub.2-R.sub.2 (SEQ ID NO: 51) represents
a peptide sequence selected from the group constituted of
SGCFGRKMDR (SEQ ID NO:33), GCFGRKMDRI (SEQ ID NO:34), CFGRKMDRIS
(SEQ ID NO:35), FGRKMDRISS (SEQ ID NO:36), FGRKMDR (SEQ ID NO:8),
SFGRKMDRISS (SEQ ID NO: 64), and CFGRKMDRISSSSGLGCK (SEQ ID NO:
65).
Preferably, the sequences different of
R.sub.1-X.sub.1-FGRKMDR-X.sub.2-R.sub.2 (SEQ ID NO: 51) are
selected from the group constituted by PRSPKMVQG (SEQ ID NO: 56),
APRSPKMV (SEQ ID NO: 57), SGLGCKVL (SEQ ID NO: 58), SPKMVQGSG (SEQ
ID NO: 59), YTLRAPRSPKMVG (SEQ ID NO: 60), YTLRAPRSPKMV (SEQ ID NO:
66), YTLRAPRSPKMVQG (SEQ ID NO: 67), SGLGCKVLRRH (SEQ ID NO: 68),
and SGLGCKVLR (SEQ ID NO: 69).
Preferably, the multiepitopic calibrators according to the
invention are selected from the group consisting of the
multiepitopic calibrators defined by the following formulae:
TABLE-US-00004 (SEQ ID NO: 66 and SEQ ID NO: 64)
Ac-YTLRAPRSPKMV-L.sub.1-SFGRKMDRISS-NH.sub.2; (SEQ ID NO: 66 and
SEQ ID NO: 65) Ac-YTLRAPRSPKMV-L.sub.1-CFGRKMDRISSSSGLGCK-NH.sub.2;
(SEQ ID NO: 56 and SEQ ID NO: 51)
Ac-YTLRAPRSPKMVQG-L.sub.1-FGRKMDR-NH.sub.2; (SEQ ID NO: 51 and SEQ
ID NO: 68) Ac-FGRKMDR-L.sub.1-SGLGC*KVLRRH-OH; (SEQ ID NO: 51 and
SEQ ID NO: 69) Ac-FGRKMDR-L.sub.1-SGLGC*KVLR-NH.sub.2; (SEQ ID NO:
59 and SEQ ID NO: 51) Ac-SPKMVQGSG-L.sub.1-FGRKMDR-NH.sub.2; (SEQ
ID NO: 66 and SEQ ID NO: 51 and SEQ ID NO: 68)
Ac-YTLRAPRSPKMV-L.sub.1-FGRKMDR-L.sub.2-SGLGC*KVLRRH-OH; and (SEQ
ID NO: 66 and SEQ ID NO: 51 and SEQ ID NO: 69)
Ac-YTLRAPRSPKMV-L.sub.1-FGRKMDR-L.sub.2-SGLGC*KVLR-NH.sub.2;
wherein Ac represents an acetyl group, and C* represents an
acetamidomethyl-blocked cysteine.
Preferably, L.sub.1 and L.sub.2 represent:
--NH--(CH.sub.2).sub.5--CO--.
This group may, in particular, derive from the coupling agent known
as hexanoic amino acid.
When E.sub.1, E.sub.2 and E.sub.3 are present, the calibrator is
said to be triepitopic, when only E.sub.1 and E.sub.2 are present,
then the calibrator is said to be biepitopic.
Moreover, the present invention also relates to a kit for detecting
human BNP(1-32) or human proBNP(1-108) as well as the respective
fragments thereof comprising the sequence FGRKMDR (SEQ ID NO: 51),
comprising at least: a ligand as defined above; and a multiepitopic
calibrator as defined above and/or a polypeptide as defined
above.
In a particular embodiment, the above kit also comprises a positive
biological control sample.
Preferably, the kit according to the invention comprises at least
the monoclonal antibody produced by the hybridoma deposited on Apr.
13, 2007 at the CNCM (Collection Nationale de Cultures de
Microorganismes, Institut Pasteur, 25, rue du Docteur Roux, 75 724
Paris Cedex 15, France) under registration number CNCM I-3746.
The following examples and figures illustrate the invention,
without limiting it.
DESCRIPTION OF THE FIGURES
FIG. 1 shows the reactivity of the 20G7 antibody with immobilised
pentadecapeptides representing the sequence of BNP(1-32) (from left
to right, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:
16 and SEQ ID NO: 17), synthesized by the Spot technique.
FIG. 2 shows the result of the alascan analysis of the binding of
20G7 antibody to FGRKMDR (SEQ ID NO: 51) epitope (substitution of
each residue of the peptide by an alanine), and shows the
importance of the F, K, and R residues.
FIG. 3 depicts the inhibition of the binding of antibodies 20G7 and
24C5 to BNP(1-32) in the presence of increasing concentrations of
soluble peptide of sequence SEQ ID NO: 51 ("AA11-AA17", diamond),
SEQ ID NO: 62 ("mutated AA11-AA17", circle) or SEQ ID NO: 9
("deleted AA11-AA17", triangle).
FIG. 4 shows the result of the alascan analysis of the binding of
11A8 antibody to FGRKMDR (SEQ ID NO: 51) epitope (substitution of
each residue of the peptide by an alanine) and shows the importance
of the F, G, K and R residues.
FIG. 5 represents a standard range of BNP(1-32) detected by the
20G7 antibody.
FIG. 6 represents a standard range of recombinant proBNP(1-108)
detected by the 20G7 antibody.
FIG. 7 shows the correlation between the detection of BNP(1-32) and
proBNP(1-108) by the 20G7 antibody in patients with congestive
heart failure.
FIGS. 8A-8C show the correlation between the detection of BNP(1-32)
and proBNP(1-108) by the 20G7 antibody in samples from subjects
with congestive heart failure of NYHA class I (FIG. 8A), NYHA class
II (FIG. 8B) and NYHA class III (FIG. 8C).
FIG. 9 shows the correlation between the detection of BNP(1-32) and
proBNP(1-108) by the 20G7 antibody, in samples from healthy
subjects.
FIG. 10 shows the correlation between the detection of BNP(1-32)
and proBNP(1-108) by the 20G7 antibody in samples from subjects
with renal failure.
FIG. 11 shows BioPlex.TM. 2200 proBNP concentrations in ischemic
stroke and control citrated plasma samples. Notched box show the
minimum, 25.sup.th, 50.sup.th, 75.sup.th percentiles and the
maximum values.
FIG. 12 shows the correlation between the detection of BNP(1-32)
and proBNP(1-108) by the 20G7 antibody in samples from subjects
with acute coronary syndrome.
FIG. 13 shows the correlation between the assay of glycosylated
proBNP(1-108) and the assay of unglycosylated proBNP(1-108) by
immunoassay using the immobilised hinge 76 antibody and in
revelation the 20G7 antibody according to the invention.
FIGS. 14 and 15 show a standard range of the biepitopic
calibrators, CaliproBNP1 and CaliproBNP3, respectively, by using
the Bioplex.TM. 2200 device.
FIG. 16 shows a standard range of the biepitopic calibrator
CaliproBNP5, proBNP(1-108) and BNP(1-32) by immunoassay using the
immobilised polyclonal antibody L21016 and in revelation the 20G7
antibody according to the invention.
FIG. 17 shows a standard range of the triepitopic calibrator
CaliproBNP6 and proBNP(1-108) in two immunoassay formats: one based
on the immobilisation of the hinge 76 antibody and in revelation
the 20G7 antibody according to the invention (open and close
circle), and the second one based on the immobilisation of the
hinge 76 antibody and in revelation an antibody directed against an
epitope localised in the C-terminal part of the BNP(1-32) (open and
close triangle).
EXAMPLES
Example 1
Peptide Synthesis
Materials and Methods:
Synthetic peptides were prepared by standard methods which are well
known to the one skilled in the art. An example of this method is
Merrifield synthesis, which is advantageous due to the fact that it
can be implemented easily (Merrifield, (1963); R. C. Sheppard
(1971); Atherton et al. (1989)). "Pioneer" synthesisers from
Perspective, or the "433A" synthesiser from ABI may be used as the
automatic synthesiser. The peptides may also be obtained by
homogenous phase synthesis.
The following syntheses were carried out in a Pioneer synthesiser
using "Fmoc" chemistry (9-fluorenylmethyloxycarbonyl): in each
step, the reagents (that is to say the protected amino acid and the
coupling activators
(TBTU(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate)/HOBt (N-hydroxybenzotriazole)) were added in
excess (in a "moles of reagent/moles of groups which can be
substituted on the resin" ratio=5). At the end of the synthesis
process, the peptide was separated from the resin by a
trifluoroacetic acid solution (reagent K). The peptide was then
precipitated in a cooled ether solution, lyophilised and then
subsequently purified by HPLC.
In this way, the inventors synthesised peptides containing the
following amino acid sequences:
TABLE-US-00005 SEQ ID NO: 4: Ac-TGCFGRKMDRISTSTAIGCKVLCys-CONH2,
SEQ ID NO: 5: 5: Ac-SGCYGRKMDRISTSTAIGCKVL-CysCONH2, SEQ ID NO: 6:
Ac-SGCFGRKMDRISSSSGLGCKVL-CysCONH2, SEQ ID NO: 7:
Ac-SGCFGRKMDRIATSTAIGCKVL-CysCONH2, SEQ ID NO: 8: Ac-FGRKMDR-CONH2,
SEQ ID NO: 9: Ac-GRKMDR-CONH2, SEQ ID NO: 10: Ac-FGRKMD-CONH2, SEQ
ID NO: 11: Ac-RKMDRI-CONH2.
Example 2
Immunogen Preparation: Coupling of a Peptide to a Carrier Protein
for Immunisation
In order to immunise mice with these peptides, it is necessary to
couple said peptides to a carrier protein such as KLH (keyhole
limpet haemocyanin), thyroglobulin, or BSA (bovine serum albumin),
via different functional groups (thiol, amine, aldehyde, etc.) so
as to render the peptide more immunogenic. The coupling reagent
used to bind the peptide to the protein may be heterobifunctional
or homobifunctional. The most frequently used reagents are BS3,
sSMCC, SPDP, glutaraldehyde, etc..
The coupling method used involved the bifunctional sSMCC (Pierce,
#22322) molecule, having an NHS ester functional group and a
maleimide group as the chemical coupling agent, and KLH (Pierce,
#77600) as the carrier protein.
2-a. KLH Activation
Method:
20 mg of KLH were solubilised in 2 ml of phosphate buffered saline
(20 mM phosphate, 0.9 M NaCl pH 7.2) in order to obtain a final
concentration of 10 mg/ml (do not vortex). In parallel, 4 mg of
sSMCC were solubilised with 400 .mu.l of water for injection to
obtain a final concentration of 10 mg/ml. 2 ml of KLH (20 mg) were
subsequently mixed with 200 .mu.l of sSMCC (2 mg), and the mixture
was incubated for 1 hour at room temperature (20.degree. C.) whilst
being stirred slowly (20 revolutions per min).
2-b. Desalting the Activated KLH:
Method:
A PD10 Sephadex TM G-25m column (Ge healthcare, USA, ref:
17-0851-01) was equilibrated with phosphate buffered saline (20 mM
phosphate, 0.9 M NaCl pH 7.2, 100 mM EDTA). The 2 ml of activated
KLH were deposited on the column, and the elution was subsequently
started with 3.5 ml of 20 mM PBS buffer supplemented with 0.9 M
NaCl, pH 7.2 and 100 mM EDTA; 500 .mu.l fractions were collected.
The optical density (OD) was measured at 280 nm for each fraction
diluted to 1/25th and the fractions containing the activated KLH
were then identified and measured in accordance with the
Beer-Lambert law: OD=.epsilon.Cl: wherein OD is the optical density
.epsilon.=1.499, C is the concentration and I=1 cm, the
concentration of the activated KLH may be determined and is reduced
to 7.4 mg/ml in phosphate buffered saline.
2-c. Coupling of the Peptide to the Activated KLH
Method:
10 mg of lyophilised peptide were solubilised in 1 ml of Milli-Q
water, which was degassed in an ultrasonic disintegrator to obtain
a final concentration of 10 mg/ml, and were then mixed with 7.4 mg
of activated KLH (i.e. 1 ml of the solution obtained in 2-b.). This
mixture was left to incubate for 2 hours at room temperature
(20.degree. C.) whilst being stirred slowly (20 rpm). A solution of
cysteine at a concentration of 5 mg/ml in a 20 mM PBS buffer+0.9 M
NaCl pH 7.2 was subsequently introduced to obtain a final
concentration of 1 mM in the peptide/KLH solution, and the entire
mixture was left to incubate for 20 minutes at room temperature
(20.degree. C.) whilst being stirred slowly (20 rpm).
2-d. Characterisation of the Coupled Peptide
Method:
The concentration of the coupled peptide was then determined by the
Bradford method (Bradford M., Anal. Biochem., 1976; 72: 248-54) as
follows: a standard range of from 50 to 1000 .mu.g/mL of KLH was
prepared in order to determine the KLH concentration of our sample
from the OD at 595 nm. In order to produce this standard range and
to carry out this assay, 50 .mu.L of each point of the sample were
diluted in 1.5 mL of Coomassie blue (Bio-Rad, #1856210).
Having determined the concentration, PBS was added to the
KLH-coupled peptide to bring the concentration of the coupled
peptide to 1 mg/mL.
Example 3
Immunisation of Mice and Production of Monoclonal Antibodies
3-a) Immunisation of Mice:
In order to produce monoclonal antibodies, ten mice (Balb/c strain
females, 5 weeks old, ref: SIFE055, Charles Rivers, Mass., USA)
were immunised using one of the following peptides:
TABLE-US-00006 (SEQ ID NO: 4) Ac-TGCFGRKMDRISTSTAIGCKVL-Cys-CONH2,
(SEQ ID NO: 5) Ac- SGCYGRKMDRISTSTAIGCKVL-CysCONH2,
coupled to KLH in accordance with Example 2 (5 mice for each
peptide).
For the first injection, an emulsion of 100 .mu.g of KLH-coupled
peptide (at a concentration of 1 mg/ml) diluted to 1/2 in Freund's
complete adjuvant (Sigma, #F-5881) was prepared, and 200 .mu.L of
said emulsion (i.e. 100 .mu.g of peptide) were injected
subcutaneously into each mouse. At intervals of 20 days, three 200
.mu.L booster shots of an emulsion of KLH-coupled peptide (i.e. 100
.mu.g of peptide) and Freund's incomplete adjuvant (Sigma, #F-5506)
were injected subcutaneously, then peritoneally, into each
mouse.
20 days after the last booster shot, and after the antibodies
obtained had been assessed by the ELISA method (in accordance with
Example 4 described below), the mouse with the greatest reaction
against BNP(1-32) was retained in order to undergo
hyperimmunisation, in accordance with the following protocol:
subcutaneous injection of 200 .mu.L of peptide-KLH at 1 mg/mL
diluted to 1/20.sup.th with PBS 45 minute-wait subcutaneous
injection of 200 .mu.L of peptide-KLH at 1 mg/mL diluted to
1/20.sup.th with PBS at a site different from the first injection
45 minute-wait subcutaneous injection of 200 .mu.L of peptide-KLH
at 1 mg/mL diluted to 1/10.sup.th with PBS at a site different from
the previous injections 30 minute-wait intraperitoneal injection of
100 .mu.L of promethazine (2.5% Phenergan, injectable solution,
UCB) diluted to 1 mg/mL with PBS 15 minute-wait intraperitoneal
injection of 200 .mu.L of peptide-KLH at 1 mg/mL diluted to
3/10.sup.ths with PBS at a site different from the previous
injections
After these immunisations, the mouse S2 immunised with the SEQ ID
NO: 4 peptide was found to produce an antiserum which was very
reactive towards BNP(1-32) when using the protocol for detecting
antibodies described below in Example 4. The lymphocytes from the
spleen of said mouse were subsequently subjected to lymphocyte
fusion, carried out in accordance with the protocol described below
in 3b.
3-b) Production of Monoclonal Antibodies:
Lymphocyte fusion of the spleen cells of the immunised mouse S2
with myeloma SP2 cells (ATCC CRL-1581) was carried out in
accordance with Kohler and Milstein's well known protocol (1975)
Nature 56:495-497.
The inventors were thus able to produce different hybrid clones. In
particular, they obtained a monoclonal antibody, which was given
the designation 20G7-15 Mar. 2007 (referred to as "20G7" in the
following for convenience). The hybridoma which secretes the
20G7-15 Mar. 2007 (20G7) monoclonal antibody was deposited at the
CNCM (French National Collection of Cultures of Microorganisms,
Institut Pasteur, 25, rue du Docteur Roux, 75 724 Paris Cedex 15,
France) with the registration number CNCM I-3746 on Apr. 13,
2007.
It goes without saying that other protocols for obtaining
monoclonal antibodies which are well known to the one skilled in
the art may be used.
Example 4
Detection of Anti-BNP(1-32) Antibodies to Assess the Response of
Mice During Immunisation
4.1 Materials: The following reagents were used: Maxisorp 96-well
flat-bottomed microplate (Nunc, Denmark) PBS (phosphate buffered
saline) buffer, pH 7.4, Gibco tablets, ref: 18912-014 (Invitrogen)
BNP(1-32): synthetic peptide (Sigma-Aldrich, USA, #B-5900) or
proBNP(1-108) (recombinant protein produced in E. Coli, HyTest,
Finland) Tween.RTM. 20 (Sigma-Aldrich, USA, #P1379) anti-mouse IgG
secondary antibody produced in rabbit and coupled to peroxidase,
(Sigma, USA, #A9044) H.sub.2O.sub.2 (0.04% in 0.1 M citrate buffer,
pH 4) OPD (ortho-phenylenediamine, Sigma, USA, #P8412) sulphuric
acid (H.sub.2SO.sub.4, 4N) serums from mice immunised in Example
3
4.2 Method and Principle:
An ELISA test was carried out on a solid support to detect the
presence of anti-BNP(1-32) antibodies in a mouse serum sample.
Some of the antigen was immobilised by adsorption in the cavities
of a 96-well microplate. After the remaining free sites were
saturated and blocked, the immune serums were left to incubate, and
the antibodies ( Ac ) which may have been present bound to the
antigen ( Ag ) and formed an Ag--Ac complex. This complex was
detected using an immunoconjugate (anti-mouse IgG antibody) coupled
to an enzyme, which in this case was HRP (horseradish peroxidase),
which transforms a colourless substrate into a coloured product
which indicates the presence of the desired antibody. The formation
of the final coloured product was quantified by carrying out an
optical density reading at 490 nm (OD). According to this method,
which is well known to the one skilled in the art, the OD obtained
indicates the presence (high OD) or absence (low OD) of antibodies
in a tested mouse serum sample. There are a number of variants of
this test (antigen capture, competition assay . . . ) which are
well known to the one skilled in the art. 1) Immobilisation of the
antigen on the microplate:
Each antigen, BNP(1-32) or proBNP(1-108), was solubilised in PBS at
a final concentration of 0.5 .mu.g/mL and was then immobilised, on
the basis of 100 .mu.L per well, on a Maxisorp microplate by being
incubated overnight at 4.degree. C. After 3 washes with PBS 0.1%
Tween.RTM. 20 (PBS-T), the microplate was saturated with a solution
(100 .mu.L/well) of 0.1% PBS-T containing 1% milk (semi-skimmed)
and was then left to incubate at 37.degree. C. for 1 hour. 2)
Immunological detection of the antibodies produced by the mice:
The microplate was washed three times with 0.1% PBS-T. Each serum
from previously immunised mice was subsequently diluted tenfold
with 0.1% PBS-T containing 0.1% milk (semi-skimmed), then deposited
on the basis of 100 .mu.L per well and left to incubate for two
hours at 37.degree. C. The microplate was again washed three times
with 0.1% PBS-T, then left to incubate for 1 hour at 37.degree. C.
in the presence of the conjugate coupled to peroxidase diluted to a
1/3,000.sup.th in 0.1% PBS-T containing 0.1% milk (semi-skimmed) on
the basis of 100 .mu.L per well. Finally, the microplate was washed
three times with 0.1% PBS-T, then the peroxidase substrate was
deposited on the basis of 100 .mu.L per well. The microplate was
placed in darkness at room temperature for 20 minutes. The
enzymatic reaction was stopped by adding 50 .mu.L of sulphuric acid
(H.sub.2SO.sub.4, 4 N) per well, and the OD at 490 nm was
subsequently measured in each well.
By using this method for detecting antibodies, the inventors found
that the serum from the mouse S2 (immunised with the SEQ ID NO: 4
peptide) was very reactive with BNP(1-32), and proBNP(1-108).
Following the lymphocyte fusion which was subsequently carried out
between the lymphocytes from said hyperimmune mouse and the Sp2
myeloma, this method also made it possible to identify a hybridoma
which produces an important monoclonal antibody: the 20G7
hybridoma, producing the 20G7 monoclonal antibody.
Example 5
Epitopic Characterisation of the 20G7 Monoclonal Antibody
5.1 Epitopic Characterisation According to the Spot Technique
5.1.1 Materials:
The equipment and reagents are all listed in C. Granier, S.
Villard, D. Laune (Mapping and Characterization of Epitopes using
the SPOT method. Cells/Cell Biology: A Laboratory Handbook, third
edition (Volume 1), chapter 62, editor: Julio Celis, Elsevier,
2005).
5.1.2 Method:
The "SPOT" or "epitope mapping" method was used to characterise the
epitope of the 20G7 monoclonal antibody. This method, described by
Frank (Tetrahedron, 1992; 48: 9217-32), allows synthesis on a
cellulose membrane of a large number of peptides with sequences
predetermined on a functionalised support
(aminopolyethyleneglycol-cellulose) and testing of their reactivity
towards a soluble ligand, which is, in the present case, the 20G7
antibody.
5.1.2.1 Peptide Synthesis
The entire peptide synthesis process (amino acid activation,
chemical reaction, etc.) is detailed in Molina et al. (Pept Res.
1996, Vol. 9: pp. 151-5), and in C. Granier, S. Villard, D. Laune
(Mapping and Characterization of Epitopes using the SPOT method.
Cells/Cell Biology: A Laboratory Handbook, third Edition (Volume
1), chapter 62, Editor: Julio Celis, Elsevier, 2005).
The BNP(1-32) sequence was synthesised entirely in the form of
overlapping pentadecapeptides (SEQ ID NO: 12 to 20), with an offset
of two amino acids:
TABLE-US-00007 SEQ ID NO: 12 SPKMVQGSGCFGRKM SEQ ID NO: 13
KMVQGSGCFGRKMDR SEQ ID NO: 14 VQGSGCFGRKMDRIS SEQ ID NO: 15
GSGCFGRKMDRISSS SEQ ID NO: 16 GCFGRKMDRISSSSG SEQ ID NO: 17
FGRKMDRISSSSGLG SEQ ID NO: 18 RKMDRISSSSGLGCK SEQ ID NO: 19
MDRISSSSGLGCKVL SEQ ID NO: 20 RISSSSGLGCKVLRR
The other following peptides were also synthesised:
TABLE-US-00008 SEQ ID NO: 21 VQGSGCFGR SEQ ID NO: 22 SPKMVQGSGC SEQ
ID NO: 23 MDRISSSSGLG SEQ ID NO: 24 RKMDRI SEQ ID NO: 25
RKMDRISS
The BNP(1-32) sequence was also synthesised in the form of
overlapping decapeptides (SEQ ID NO: 26 to 48), with an offset of 1
amino acid:
TABLE-US-00009 SEQ ID NO: 26 SPKMVQGSGC SEQ ID NO: 27 PKMVQGSGCF
SEQ ID NO: 28 KMVQGSGCFG SEQ ID NO: 29 MVQGSGCFGR SEQ ID NO: 30
VQGSGCFGRK SEQ ID NO: 31 QGSGCFGRKM SEQ ID NO: 32 GSGCFGRKMD SEQ ID
NO: 33 SGCFGRKMDR SEQ ID NO: 34 GCFGRKMDRI SEQ ID NO: 35 CFGRKMDRIS
SEQ ID NO: 36 FGRKMDRISS SEQ ID NO: 37 GRKMDRISSS SEQ ID NO: 38
RKMDRISSSS SEQ ID NO: 39 KMDRISSSSG SEQ ID NO: 40 MDRISSSSGL SEQ ID
NO: 41 DRISSSSGLG SEQ ID NO: 42 RISSSSGLGC SEQ ID NO: 43 ISSSSGLGCK
SEQ ID NO: 44 SSSSGLGCKV SEQ ID NO: 45 SSSGLGCKVL SEQ ID NO: 46
SSGLGCKVLR SEQ ID NO: 47 SGLGCKVLRR SEQ ID NO: 48 GLGCKVLRRH
A selection of heptapeptides with an offset of one amino acid were
also synthesised (SEQ ID NO: 49 to 53):
TABLE-US-00010 SEQ ID NO: 49 GCFGRKM SEQ ID NO: 50 CFGRKMD SEQ ID
NO: 51 FGRKMDR SEQ ID NO: 52 GRKMDRI SEQ ID NO: 53 RKMDRIS
5.1.2.2 Immunological Test
The followed test for immunoreactivity has been described in detail
in Laune et al. (J. Immunol. Methods, 2002, Vol. 267(1), pp.
53-70). In short, the principle was as follows. The membrane was
rehydrated by three TBS baths (tris-buffered saline, pH 7.0) with a
duration of 10 minutes in each case, and was subsequently saturated
by being incubated overnight at room temperature, whilst being
stirred, in the presence of 15 ml of a 10% saturation buffer
("blocking buffer", Roche) and 5% saccharose in TBS 0.1% Tween.RTM.
20 (TBS-T). After the membrane was washed three times for 10
minutes with 0.1% TBS-T, the membrane was left to incubate for 90
minutes at 37.degree. C. whilst being stirred, in the presence of
the antibody to be tested (20G7 in this case) and the conjugate
coupled to alkaline phosphatase diluted with the saturation buffer.
After washing the membrane twice with 0.1% TBS-T, then twice with
CBS (citrate buffered saline), each bath lasting 10 minutes, the
alkaline phosphatase substrate was added and the membrane was
incubated at room temperature for 1 to 30 minutes, depending on the
speed at which the signal appeared.
5.1.2.3 Results
In the present case, the BNP(1-32) sequence was synthesised
entirely in the form of overlapping pentadecapeptides (SEQ ID NO:
12 to 20), with an offset of two amino acids. As shown in FIG. 1,
when the peptides are contacted with the purified 20G7 antibody,
only five successive peptides react with the antibody, and the
common sequence thereof is F.sub.11GRKMDR.sub.17 (SEQ ID NO: 51)
(FIG. 1):
TABLE-US-00011 SEQ ID NO: 13 K M V Q G S G C F G R K M D R SEQ ID
NO: 14 V Q G S G C F G R K M D R I S SEQ ID NO: 15 G S G C F G R K
M D R I S S S SEQ ID NO: 16 G C F G R K M D R I S S S S G SEQ ID
NO: 17 F G R K M D R I S S S S G L G
To ensure that only this pattern is actually involved in the
binding of the antibody to BNP(1-32), shorter peptides
(decapeptides (SEQ ID NOs: 26 to 48) and heptapeptides (SEQ ID NOs:
49 to 53) were also synthesised, with an offset of only one amino
acid, in order to confirm and validate the epitope. In each
experiment, the common peptide sequence identified by 20G7 was
F.sub.11GRKMDR.sub.17 (SEQ ID NO: 51) (for decapeptides, SEQ ID NO:
33 to 36, and for heptapeptides, SEQ ID NO: 51).
In order to determine which residues are critical and essential for
the recognition of the epitope, each residue of the minimal
sequence F.sub.11GRKMDR.sub.17 (SEQ ID NO: 51) was substituted
successively by an alanine (A) in order to assess the involvement
of each individual residue in accordance with the "Alascan" method,
which is well known and described (Laune et al., above). As shown
in FIG. 2, binding decreased to 82%, 95% and 85% when the F.sub.11,
K.sub.14 and R.sub.17 residues were respectively substituted by an
alanine, which indicates that these residues are essential.
These amino acids in positions 11, 14 and 17 are essential for the
recognition of the epitope by the 20G7 monoclonal antibody. These
data are the mean values of a plurality (n=4) of repeat
experiments. For this reason, it is therefore clear that the
F.sub.11GRKMDR.sub.17 (SEQ ID NO: 51) epitope, comprising the
essential F.sub.11, K.sub.14 and R.sub.17 amino acids, according to
the invention, is different from that recognised in the patent
application WO2006/88700 which discloses another epitope
(R.sub.13(K.sub.14)(M.sub.15)D.sub.16R.sub.17I.sub.18) (SEQ ID NO:
24), the important amino acids of which are R.sub.13, D.sub.16,
R.sub.17 and I.sub.18.
In order to achieve a better understanding of the contributory
effect of these residues to the binding process of the 20G7
antibody, the F.sub.11, K.sub.14 and R.sub.17 amino acids were
substituted by amino acids with closed biochemical properties. For
example, F.sub.11 was substituted by other aromatic amino acids
(tryptophan and tyrosine). The fact that the sequence composed of
these "homologous" amino acids was recognised in the same way by
the 20G7 antibody suggests that it is the aromatic nature of the
peptide in position 11 that is essential for the antibody binding.
With regard to K.sub.14 and R.sub.17, they were both substituted by
an arginine and a lysine to study the effect of the lateral chain
of the amino acid, and also the presence of a positive charge. It
was also found in this case that the substitution was effectively
conservative, since binding to 20G7 was retained, thus underlining
the importance of the positive charge.
The same does not apply to the 24C5 antibody from HyTest, which has
different essential residues.
The VQGSGCFGR, SPKMVQGSGC, MDRISSSSGLG, R.sub.13KMDRI.sub.18 and
R.sub.13KMDRISS.sub.20 (SEQ ID NO: 21 to SEQ NO: 25) peptide
sequences of human BNP(1-32), which were also synthesised on a
membrane, were tested with the 20G7 antibody using the Spot method.
Since residues which are essential for the binding of 20G7 were
absent, said antibody did not bind to said peptides at all, with
the result that 20G7 exhibited a cross reaction of less than 2%
with these peptides.
5.2 Characterisation with Soluble Peptides
In a second step, to ensure that the F.sub.11GRKMDR.sub.17 (SEQ ID
NO: 51) peptide was really the epitope of the 20G7 antibody, this
sequence was synthetized in a soluble form in order to carry out
competition assays between this peptide and BNP(1-32). Parallely,
in order to confirm the high contribution of the F.sub.11 residue
in the binding to the 20G7 antibody, two other additional peptides
(one wherein F was substituted by A, and the other wherein F was
simply deleted) were also synthetised in a soluble form. Moreover,
similar competition assays were carried out with the 24C5 antibody
of Hytest to demonstrate that the importance of this residue is
specific of the 20G7 antibody.
1-SEQ ID NO 51: sequence of the native epitope:
F.sub.11GRKMDR.sub.17
2-SEQ ID NO 62: mutated sequence of the epitope:
A.sub.11GRKMDR.sub.17
3-SEQ ID NO 9: sequence deleted of the F.sub.11 residue:
G.sub.12RKMDR.sub.17
5.2.1 Materials Maxisorp 96-well flat-bottomed microplate (Nunc,
Denmark) PBS (phosphate buffered saline) buffer, pH 7.4, Gibco
tablets, ref: 18912-014 (Invitrogen) BNP(1-32): synthetic peptide
(Sigma-Aldrich, USA, #B-5900) Tween.RTM. 20 (Sigma-Aldrich, USA,
#P1379) monoclonal 20G7 antibody (Bio-Rad) monoclonal 24C5 antibody
(HyTest, Turku, Finland) anti-mouse IgG secondary antibody produced
in rabbit and coupled to peroxidase, (Sigma, USA, #A9044)
H.sub.2O.sub.2 (0.04% in 0.1 M citrate buffer, pH 4) OPD
(ortho-phenylenediamine, Sigma, USA, #P8412) sulphuric acid
(H.sub.2SO.sub.4, 4N)
5.2.2 Methods
The principle of the immunoassay was the same as the one described
in 4.2. Briefly, synthetic BNP(1-32) was diluted in PBS buffer to
be directly immobilised on a Maxisorp microplate at 0.5 .mu.g/ml. A
standard range from 20 to 10,000 ng/ml of each soluble peptide
AA11-AA17 (native, mutated or deleted) was prepared in buffer/serum
and mixed to 100 .mu.l of monoclonal antibody solution (20G7 or
24C5 antibody), at a final concentration of 0.5 .mu.g/ml in PBS
0.1% Tween.RTM. 20 (PBS-T) containing 0.1% milk (semi-skimmed). The
binding of the antibody was then detected by an anti-mouse
conjugate labelled with peroxydase. The intensity of the response
of the 20G7 antibody was compared to the one of monoclonal 24C5
antibody from HyTest. The percentage of inhibition corresponding to
the decrease of the recognition of BNP(1-32) in the presence of the
soluble peptide was determined for each soluble peptide, in order
to determine 1) that the sequence F.sub.11GRKMDR.sub.17 (SEQ ID NO:
51) effectively represents the 20G7 antibody epitope and 2) that
the F.sub.11 residue was essential for the recognition of BNP(1-32)
by 20G7.
5.2.3 Results
FIG. 3 depicts the percentage of inhibition of the binding of the
monoclonal antibody (20G7 or 24C5) to BNP(1-32) in the presence of
increasing concentrations of soluble peptides (native: SEQ ID NO:
51, mutated: SEQ ID NO: 62, deleted: SEQ ID NO: 9).
It is highly remarkable to note the 20G7 antibody of the present
invention behaves distinctly from the 24C5 antibody in the
recognition of BNP(1-32) in the presence of the soluble AA11-AA17
peptide mutated (SEQ NO:62) or deleted (SEQ NO:9). Adding soluble
peptide mutated (A.sub.11GRKMDR.sub.17(SEQ ID NO: 62)) or deleted
(G.sub.12RKMDR.sub.17) (SEQ ID NO: 9) does not inhibit the
recognition of BNP(1-32) by 20G7, whatever the added peptide
concentration (up to 20 .mu.g/ml), whereas a total inhibition is
observed when the native peptide (F.sub.11GRKMDR.sub.17
corresponding to the sequence SEQ ID NO: 51) is added. This
experiment confirms the importance of the F.sub.11 residue in the
binding of the 20G7 antibody to BNP(1-32), on the contrary to the
24C5 antibody.
Example 6
Epitopic Characterisation of Other Anti-BNP(1-32) Monoclonal
Antibodies--Comparison with the 20G7 Antibody
6.1. Materials, Methods and Protocols:
The same nitrocellulose membranes and the same conditions of
reactivity as those described in Example 5 were used to study these
antibodies.
6.2. Results:
The inventors thus obtained a series of following characterized
monoclonal antibodies: 20G7, 11A8, 17F10, Mab1, Mab2 and Mab3. As
shown in Table 1, the other monoclonal antibodies have the same
epitope as the monoclonal 20G7 of the present invention, i.e.
FGRKMDR (SEQ ID NO: 51), but have different essential amino acids
compared to 20G7.
TABLE-US-00012 TABLE 1 Characteristics of the monoclonal antibodies
Monoclonal Epitope Essential antibody Immunogen (SEQ ID NO: 51)
residues 20G7 SEQ ID NO: 4 F.sub.11GRKMDR.sub.17 F, K and R 11A8
SEQ ID NO: 4 F.sub.11GRKMDR.sub.17 F, G, K and R 17F10 SEQ ID NO: 4
F.sub.11GRKMDR.sub.17 F, K, D and R Mab1 SEQ ID NO: 4
F.sub.11GRKMDR.sub.17 F, G, R and K Mab2 SEQ ID NO: 4
F.sub.11GRKMDR.sub.17 F and K Mab3 SEQ ID NO: 4
F.sub.11GRKMDR.sub.17 F, G, R and K
FIG. 4 shows for example the result of the alascan analysis
(successive substitution of each residue of the sequence
F.sub.11GRKMDR.sub.17 (SEQ ID NO: 51) by an alanine to assess the
individual implication of each residue, method described above in
5.1.2.3) for 11A8 antibody. A substitution of the four residues (F,
G, K and R) results in a significant loss of binding of the
sequence F.sub.11GRKMDR.sub.17 (SEQ ID NO: 51), demonstrating that
they are essential for the binding to BNP(1-32).
Example 7
Characterisation of the Antibody-Antigen Interaction of the
Monoclonal Antibodies by Surface Plasmon Resonance Technology
7.1. Materials: BIAcore.RTM. 2000 & 3000 analyser (Pharmacia,
Uppsala, Sweden) BNP(1-32) (synthetic peptide, Sigma, USA, #B-5900)
proBNP(1-108) (recombinant protein produced in E. Coli, HyTest,
Finland) anti Fc fragment antibodies (Sigma, USA) monoclonal
antibodies 20G7, 11A8, 17F10 (Bio-Rad, Marnes la Coquette, France)
PBS buffer (phosphate buffered saline), pH 7.4
7.2. Method:
7.2.1. Principle:
The BIAcore.RTM. 2000 & 3000 analyser (the principle of which
is based on the surface plasmon resonance technology (SPR)), was
used to define the kinetics and the affinity of the interaction of
the 20G7 monoclonal antibody and other monoclonal antibodies with
BNP(1-32) or proBNP(1-108). The inventors followed the
manufacturer's instructions.
The surface plasmon resonance SPR technique (BIAcore.RTM.,
Pharmacia) was described in its entirety in Ferrieres et al. (2000,
FEBS Letters, 479(3): 99-105). A monoclonal antibody was
immobilised on a biosensor or a solid surface by using an anti Fc
fragment antibody whilst the soluble antigen (BNP(1-32) or
proBNP(1-108)) circulated at increasing concentrations (0.001256 to
0.125 .mu.g/ml) in a constant flow on the surface of the biosensor
at room temperature. The angle at which the SPR signal is detected
is directly proportional to the refractive index of the medium in
which the evanescent wave propagates. The variations in the
refractive index are expressed in resonance units (RU, where 1000
resonance units correspond to 1 ng of fixed proteins per mm.sup.2
of active area). The quantification of the interaction and the
affinity between the antigen and the monoclonal antibody is
assessed by calculating the association rate constant (ka) and the
dissociation rate constant (kd) by global data processing using the
manufacturer's software BIAevaluation (BIAcore.RTM., Pharmacia,
Uppsala, Sweden). The equilibrium dissociation constant (KD=kd/ka)
in mol/l reflects the affinity of the BNP(1-32) or proBNP(1-108)
antigen for the monoclonal antibody.
7.2.2. Results:
Table 2 shows the characteristics of the interaction between the
monoclonal anti-BNP antibodies (including 20G7) and the two
recombinant antigens BNP(1-32) and proBNP(1-108).
TABLE-US-00013 TABLE 2 Interactions between various monoclonal
anti-BNP antibodies and the BNP(1-32) and proBNP(1-108) antigens.
BNP ka (M.sup.-1 s.sup.-1) kd (s.sup.-1) KA (M.sup.-1) KD (M) 20G7
1.40 10.sup.6 2.38 10.sup.-4 5.90 10.sup.9 1.70 10.sup.-10 11A8
8.58 10.sup.5 2.23 10.sup.-3 3.85 10.sup.8 2.59 10.sup.-9 17F10
5.84 10.sup.5 2.82 10.sup.-4 2.07 10.sup.9 4.83 10.sup.-10 ProBNP
ka (M.sup.-1 s.sup.-1) kd (s.sup.-1) KA (M.sup.-1) KD (M) 20G7 1.02
10.sup.6 1.74 10.sup.-4 5.90 10.sup.9 1.69 10.sup.-10 11A8 7.63
10.sup.5 1.19 10.sup.-3 6.43 10.sup.8 1.56 10.sup.-9 17F10 9.34
10.sup.5 2.15 10.sup.-4 4.35 10.sup.9 2.30 10.sup.-10
Table 2 summarises the different characteristics (association rate
constant (ka) and dissociation rate constant (kd) values allowing
the equilibrium dissociation constant (KD in M) of the interaction
between BNP(1-32) or proBNP(1-108) and the monoclonal antibodies to
be calculated. These results for the interaction confirm the data
obtained with the BNP(1-32) and proBNP(1-108) assays inasmuch as
the 20G7 monoclonal antibody exhibits an excellent association
constant (ka) and a low dissociation constant (kd), allowing it to
be characterised by an excellent affinity constant of 1.70.sup.-10
M, identical for BNP(1-32) and proBNP(1-108) (Table 2).
Examples are also provided for other monoclonal antibodies (11A8
and 17F10), the affinity constants of which are in the nanomolar
range (2.times.10.sup.-10 to 9.35.times.10.sup.-10 M, Table 2).
Example 8
BNP(1-32) Assay Using the 20G7 Monoclonal Antibody
8.1. Materials: Maxisorp 96-well flat-bottomed microplate (Nunc,
Denmark) PBS (phosphate buffered saline) buffer, pH 7.4, Gibco
tablets, ref: 18912-014 (Invitrogen) BNP(1-32) synthetic peptide
(Sigma-Aldrich, USA, #B-5900) proBNP(1-108) (recombinant protein
produced in E. Coli, HyTest, Finland) Tween.RTM. 20 (Sigma-Aldrich,
USA, #P1379) L21016 rabbit polyclonal antibody obtained by
immunising rabbits with an immunogen targeting the 1-10 region of
BNP(1-32), its epitope being the sequence S.sub.1PKMV.sub.5 (SEQ ID
NO: 54) of BNP(1-32) 20G7 monoclonal antibody (Bio-Rad) 24C5 and
26E2 monoclonal antibodies (HyTest, Turku, Finland) anti-mouse IgG
antibody conjugate produced in rabbit and coupled to peroxidase,
(Sigma, USA, #A9044) 0.04% H.sub.2O.sub.2 in a 0.1 M citrate
buffer, pH 4 OPD (ortho-phenylenediamine, Sigma, USA, #P8412)
sulphuric acid (H.sub.2SO.sub.4, 4N)
8.2. Method and Principle:
Initially, a standard range of 20 to 10,000 pg/ml of BNP(1-32) was
prepared in a buffer/serum from synthetic BNP(1-32).
The assay was based on the sandwich ELISA principle on a
microplate, using the L21016 rabbit polyclonal antibody (Bio-Rad)
for capture on a solid phase, its epitope being the sequence
S.sub.1PKM V.sub.5 of BNP(1-32) fixed by passive adsorption by way
of 100 .mu.l of 5 .mu.g/ml solution per well.
100 .mu.l of monoclonal antibody solution (20G7, 24C5 or 26E2
antibodies) in solution at a concentration of 0.5 .mu.g/ml in PBS
0.1% Tween.RTM. 20 (PBS-T) buffer containing 0.1% milk
(semi-skimmed) were used as detection reagents. The intensity of
the response of 20G7 was thus compared to that of the 24C5 and 26E2
monoclonal antibodies.
Table 3 summarises the results of the analytical assay of
BNP(1-32), expressed in optical density (OD) at 490 nm and obtained
by said antibodies in the presence of standard concentrations of
BNP(1-32).
TABLE-US-00014 TABLE 3 OD values obtained during the analytical
assay of BNP(1-32) with different antibodies BNP(1-32) (pg/ml) 20G7
24 C5 26 E2 10,000 3.752 0.077 0.048 5,000 3.056 0.068 0.041 2,500
1.950 0.067 0.035 1,250 1.111 0.056 0.031 625 0.625 0.059 0.029
312.5 0.404 0.063 0.027 156.25 0.180 0.071 0.032 78 0.099 0.052
0.055 39 0.066 0.055 0.032 20 0.031 0.068 0.034 0 0.024 0.071
0.022
It is highly remarkable to note that the two antibodies 24C5 and
26E2 behave quite differently from the 20G7 antibody of the present
invention. These results thus confirm that in the latter assay
format, 20G7 is much more suitable than the 24C5 and 26E2
antibodies for BNP(1-32) assay.
The standard range shown in FIG. 5 and obtained with the 20G7
monoclonal antibody is linear from 20 to 10,000 pg/ml
(r.sup.2=0.96). The two commercial antibodies 24C5 and 26E2 are not
very effective or not at all effective in detecting BNP(1-32), even
at high concentrations of the analyte (Table 3).
Example 9
Study of the Complementarity of Monoclonal Antibodies in the
Sandwich ELISA
9.1. Materials: Maxisorp 96-well flat-bottomed microplate (Nunc,
Denmark) primed by the L21016 rabbit polyclonal antibody (Bio-Rad)
which recognises the sequence S.sub.1PKM V.sub.5 (SEQ ID NO: 54) of
BNP(1-32) PBS (phosphate buffered saline) buffer, pH 7.4, Gibco
tablets, ref: 18912-014 (Invitrogen) Tween.RTM.20 (Sigma-Aldrich,
USA, #P1379) BNP(1-32) synthetic peptide (Sigma-Aldrich, USA,
#B-5900) proBNP(1-108) (recombinant protein produced in E. Coli,
HyTest, Finland) 20G7 antibody (directed against the epitope
F.sub.11GRKMDR.sub.17 (SEQ ID NO: 8) prepared in different
concentrations from 0.001 to 1 .mu.g/ml 50B7 monoclonal antibody
(HyTest, Finland) which recognises the C-terminal portion of
BNP(1-32) at a single concentration of 0.5 .mu.g/ml anti-mouse IgG
secondary antibody produced in rabbit and coupled to peroxidase,
(Sigma, USA, #A9044) 0.04% H.sub.2O.sub.2(in 0.1 M citrate buffer,
pH 4, Sigma, USA) OPD (ortho-phenylenediamine, Sigma, USA, #P8412)
sulphuric acid (H.sub.2SO.sub.4, 4N)
9.2. Method:
9.2.1. Principle:
The assay was based on the sandwich ELISA principle on a
microplate, using the L21016 rabbit polyclonal antibody (Bio-Rad)
for capture on a solid phase, its epitope being the sequence
S.sub.1PKMV.sub.5 (SEQ ID NO: 54) of BNP(1-32) fixed by passive
adsorption (see Example 8), and a combination of two monoclonal
antibodies (the 20G7 monoclonal antibody directed against the
epitope F.sub.11GRKMDR.sub.17 (SEQ ID NO: 8) and the 50B7
monoclonal antibody (HyTest, Finland) which targets the C-terminal
portion of BNP(1-32)) for detection. However, the 20G7 antibody was
used at variable concentrations while the 50B7 monoclonal antibody
was used at a constant concentration of 0.5 .mu.g/ml.
The epitopic complementarity of the 20G7 and 50B7 antibodies was
studied at variable concentrations of the 20G7 antibody. This
format allowed the cooperativity of the two monoclonal antibodies
to be assessed in order to improve the BN P(1-32) detection.
9.2.2. Protocol:
100 .mu.l of 5 ng/ml BNP(1-32) solution was added into each
microplate well, in which the L21016 polyclonal antibody was
adsorbed, and was left to incubate for two hours at 37.degree. C.
The microplate was washed three times with 0.1% PBS-T, then 100
.mu.l of a mixture containing the 50B7 antibody and one of the 20G7
antibody dilutions were distributed thereon, and it was left to
incubate for 2 hours at 37.degree. C. After three washes with 0.1%
PBS-T, the peroxidase--rabbit anti-mouse IgG antibody conjugate
(diluted to a 1/3,000th with 0.1% PBS-T and containing 0.1% milk
(semi-skimmed), on the basis of 100 .mu.l per well, was left to
incubate for 1 hour at 37.degree. C. Finally, after 3 washes with
0.1% PBS-T, the H.sub.2O.sub.2+OPD solution was deposited on the
basis of 100 .mu.L/well. The microplate was placed in darkness at
room temperature for 20 minutes. The enzymatic reaction was stopped
by adding 50 .mu.L of sulphuric acid (H.sub.2SO.sub.4, 4 N) per
well, and the OD at 490 nm was subsequently measured in each
well.
9.2.3. Results
Table 4 shows the results of the analytical assay of BNP using the
20G7 and 50B7 monoclonal antibodies, expressed in optical density
at 490 nm.
TABLE-US-00015 TABLE 4 Cooperativity of the two monoclonal
antibodies for detecting BNP(1-32) 20G7 range Optical Density
(.mu.g/ml) 490 nm 0.5 3.792 0.1 3.753 0.05 3.747 0.01 3.531 0.005
3.272 0.001 1.948 0 1.296
It was noted that there is synergism between the two antibodies. In
other words, it was noted that the effects of the two antibodies
were added: in the absence of 20G7, the signal (OD) was limited to
1.296, and the more 20G7 was added, the more the resulting signal
(OD) increased.
By using both the 20G7 monoclonal antibody, which targets the
F.sub.11GRKMDR.sub.17 (SEQ ID NO: 8) epitope according to the
invention (located in the loop of BN P(1-32)), and the 50B7
monoclonal antibody, which targets the C-terminal region of
BNP(1-32), BNP(1-32) detection is significantly improved. This
demonstrates the cumulative or cooperative contribution of the two
monoclonal antibodies used in detection.
This kind of complementarity may also be envisaged between the 20G7
monoclonal antibody and other antibodies which recognise an epitope
located in other positions in the BNP(1-32) sequence (principally
in the N-terminal position, C-terminal position). The number of
antibodies used may also be greater than two as long as no steric
hindrance problems are encountered.
Example 10
proBNP(1-108) Assay Using the 20G7 Monoclonal Antibody
10.1. Materials: BNP(1-32) synthetic peptide (Sigma-Aldrich, USA,
#B-5900) proBNP(1-108) (recombinant protein produced in E. Coli,
HyTest, Finland). Maxisorp 96-well flat-bottomed microplate (Nunc,
Denmark) primed with monoclonal antibodies (hinge 76 antibody, for
example, from Bio-Rad) which recognises the proBNP(1-108) hinge
sequence: epitope RAPR.sub.76S.sub.77P (SEQ ID NO: 55) (Giuliani et
al., Clin. Chem., 52: 6, 1054-1061, 2006) 20G7 monoclonal antibody
(Bio-Rad) coupled to peroxidase 24C5 monoclonal antibody (HyTest,
Finland) coupled to peroxidase 26E2 monoclonal antibody (HyTest,
Finland) coupled to peroxidase PBS (phosphate buffered saline)
buffer, pH 7.4, Gibco tablets, ref: 18912-014 (Invitrogen)
Tween.RTM.20 (Sigma-Aldrich, USA, #P1379)
10.2. Method:
10.2.1. Principle of the Analytical Assay of proBNP(1-108):
Initially, a standard range of 20 to 10,000 pg/ml of proBNP(1-108)
was prepared in a 0.1% PBS-T buffer from recombinant
ProBNP(1-108).
The assay is based on the sandwich ELISA principle on a microplate,
using for capture on a solid phase a monoclonal antibody (hinge 76
antibody, for example, from Bio-Rad), and recognising the hinge
sequence of proBNP(1-108): epitope RAPR.sub.76S.sub.77P (SEQ ID NO:
55) fixed by passive adsorption by way of 100 .mu.l of 0.5 .mu.g/ml
solution per well.
100 .mu.l of monoclonal antibody solution (20G7, 24C5 or 26E2
antibodies) at a concentration of 0.5 .mu.g/ml and coupled to
peroxidase in solution in 0.1% PBS-T buffer (containing 0.1%
semi-skimmed milk) were used as detection antibodies.
Besides this technical point, the protocol was identical to that of
the ELISA in Example 8. The detection characteristics of 20G7 were
thus compared with those of the 24C5 and 26E2 monoclonal
antibodies.
Table 5 shows the results of the analytical assay of proBNP(1-108)
which are expressed in optical density (OD) and were obtained by
using said antibodies in the presence of standard concentrations of
proBNP(1-108).
10.3. Results:
TABLE-US-00016 TABLE 5 Analytical assay of proBNP(1-108) with
different antibodies ProBNP(1-108) 24 C5 26 E2 (pg/ml) 20G7
(Hytest) (Hytest) 10,000 >4 0.220 0.158 5,000 3.845 0.117 0.041
2,500 3.272 0.069 0.031 1,250 1.955 0.068 0.024 625 0.997 0.070
0.024 312.5 0.512 0.035 0.028 156.25 0.264 0.055 0.027 78 0.126
0.056 0.033 39 0.092 0.056 0.028 20 0.069 0.052 0.028 0 0.045 0.044
0.024
It is highly remarkable to note that the 20G7 antibody of the
present invention detects not only BNP(1-32) but also
proBNP(1-108). Moreover, in this case too, the two HyTest
antibodies behave quite differently from the 20G7 antibody. This
confirms the significant benefit of the 20G7 antibody in BNP(1-32)
and proBNP(1-108) assays.
FIG. 6 illustrates the linear standard range, obtained with the
20G7 monoclonal antibody, of 20 to 10,000 pg/ml of proBNP
(r.sup.2=0.99, FIG. 6), whilst the two commercial antibodies from
Hytest are not very effective or not effective at all in detecting
proBNP(1-108), even at high proBNP(1-108) concentrations (Table
5).
Example 11
proBNP(1-108) and BNP(1-32) Assays Using Other Monoclonal
Antibodies Obtained by the Inventors
Table 6 shows the results produced, in accordance with the two
ELISA protocols from examples 8 and 10, with the 11A8 and 17F10
monoclonal antibodies.
These labelled monoclonal antibodies used in detection are highly
capable of detecting BNP(1-32) and proBNP(1-108) when a rabbit
polyclonal (L21016) which targets the .sup.1SPKMV.sup.5 (SEQ ID NO:
54) region or the hinge 76 antibody respectively, are used for
capture.
Table 6 shows the results of the analytical assays of proBNP(1-108)
and BNP(1-32) which are expressed in optical density and were
obtained by said antibodies in the presence of standard
concentrations of proBNP(1-108) and BNP(1-32) respectively.
TABLE-US-00017 TABLE 6 Analytical assay of proBNP(1-108) and
BNP(1-32) with different monoclonal antibodies BNP(1-32) or
proBNP(1- proBNP(1-108) BNP(1-32) 108) (pg/ml) 17F10 11A8 17F10
11A8 10,000 3.769 3.734 3.717 3.693 5000 3.808 3.779 2.712 2.534
2,500 3.453 3.024 1.495 1.481 1,250 2.118 1.406 0.828 0.871 625
0.937 0.618 0.341 0.389 312.5 0.489 0.259 0.172 0.211 156 0.242
0.130 0.095 0.123 78 0.139 0.088 0.070 0.114 39 0.125 0.087 0.059
0.097 20 0.100 0.078 0.059 0.071 0 0.076 0.063 0.059 0.068
It is highly remarkable to note that the 17F10 and 11A8 antibodies
of the present invention detect not only BNP(1-32) but also
proBNP(1-108).
Example 12
BNP(1-32) Assay and proBNP(1-108) Assay in Subjects with Congestive
Heart Failure and in Normal Subjects
12.1. Samples: 55 EDTA plasmas from subjects with congestive heart
failure, who belonged to one of NYHA (New York Heart Association)
classes Ito III and had signed a voluntary consent form,
originating from a commercial source (ProMedex, NY, USA). The
studied population is as follows: 10 patients of NYHA class I, 21
patients of NYHA class II and 24 patients of NYHA class III. 48
EDTA plasmas from normal subjects (healthy volunteers, ProMedex,
NY, USA).
12.2. Materials and Methods for the BNP(1-32) and the proBNP(1-108)
Assays
The materials and methods used were identical to those described
above in 8.2 for BNP(1-32), and to those described above in 10.2
for the proBNP(1-108).
12.3 Results
12.3.1. Results of the BNP(1-32) and the proBNP(1-108) Assays in
Patients with Congestive Heart Failure
The BNP(1-32) values obtained from plasmas from patients with
congestive heart failure, by means of the BNP(1-32) assay disclosed
in 8.2, were found to correlate with those of the proBNP(1-108)
assay according to the invention (r.sup.2=0.935, FIG. 7).
More in detail, correlations are maintained when the patients are
studied according to their NYHA class (r.sup.2=0.997,
r.sup.2=0.903, r.sup.2=0.832 respectively for the NYHA classes I,
II and III, FIGS. 8 A, B and C, respectively).
Thus, these results with the 20G7 antibody confirm once more the
usefulness of the BNP(1-32) or the proBNP(1-108) assay as a marker
of congestive heart failure.
These experiments were reproduced with the 24C5 and 26E2 antibodies
(HyTest) in a labelled form, but no correlation was observed.
12.3.2. Results of the BNP(1-32) and the proBNP(1-108) Assays in
Healthy Subjects
The proBNP(1-108) values obtained from plasmas from healthy
subjects, by means of the proBNP(1-108) assay using the monoclonal
hinge 76 antibody in the solid phase and the 20G7
antibody-peroxidase conjugate for detection, were found to be
highly correlated (r.sup.2=0.702) with those of the BNP(1-32) assay
using the 20G7 antibody in detection (FIG. 9).
In conclusion, it is therefore very clear that the 20G7 antibody
according to the invention is entirely appropriate for BNP(1-32)
and proBNP(1-108) assays in patients suffering from congestive
heart failure, by detecting a higher amount of BNP(1-32) and
proBNP(1-108) in patients suffering from congestive heart failure
than in healthy subjects (Table 7).
TABLE-US-00018 TABLE 7 proBNP(1-108) (pg/ml) BNP(1-32) (pg/ml)
Healthy subjects 37 .+-. 32 227 .+-. 172 NYHA patients 762 .+-. 839
1716 .+-. 1754
Example 13
BNP(1-32) and proBNP(1-108) Assays in Patients Suffering from Renal
Failure
13.1. Samples:
EDTA plasmas from 33 patients with renal failure who has signed a
voluntary consent form, originating from Lapeyronie hospital,
Montpellier, France.
13.2. Materials and Methods for the BNP(1-32) and the proBNP(1-108)
Assays
The materials and methods used were identical to those described
above in 8.2 for BNP(1-32) and in 10.2 for proBNP(1-108).
13.3 Results
The proBNP(1-108) values obtained from plasmas of patients
suffering of renal failure, by means of the proBNP(1-108) assay
using the hinge 76 antibody in the solid phase and 20G7
antibody-peroxydase conjugate in detection, were found to be
strongly correlated (r.sup.2=0.899) to those of the BNP(1-32) assay
using the 20G7 antibody according to the invention (FIG. 10). The
20G7 antibody according to the invention is highly appropriate for
the BNP(1-32) and the proBNP(1-108) assay in patients with renal
failure.
Example 14
proBNP(1-108) Assay in Patients with Ischemic Stroke
14.1. Patients Samples: 32 citrated plasma samples from patients
with ischemic stroke admitted to the Emergency Department within 3
hours of the Stroke onset were tested. The stroke severity was
assessed by the National Institutes of Health Stroke Scale (NIHSS).
42 citrated plasma samples from apparently healthy blood donor
matched by gender and age with the patients from the Stroke
population were tested. All the citrated plasma samples were stored
at -80.degree. C. Prior the analysis, the samples were thawed and
centrifuged at 3000 g for 15 min at 4.degree. C.
14.2. Material and Method:
All the samples were tested with the BioPlex.TM. 2200 proBNP assay
(Bio-Rad).
14.2.1. Principe of the Technology:
The BioPlex.TM. 2200 combines multiplex, magnetic bead and flow
cytometry technologies to provide multi-analyte detection on a
fully automated random access platform. Magnetic particles (8 .mu.m
diameter, carboxyl-modified surface) are dyed with two fluorophores
(classification dyes, CL1 and CL2) which emit at distinct
wavelengths and adsorb significantly at 635 nm. The reporter
fluorophore, .beta.-phycoerythrin (PE) was chosen for its high
molar extinction coefficient, quantum yield, resistance to
photobleaching, lack of self-quenching and stability. The detector
simultaneously measures light at three wavelengths: the two
classification dyes and the reporter dye.
14.2.2. BioPlex.TM. 2200 proBNP: Assay Principle
The BioPlex.TM. 2200 proBNP assay is a two-step sandwich
fluorescence immunoassay. In a first step, the BioPlex.TM. 2200
system combines 50 .mu.L of patient sample, magnetic dyed beads
coated with the anti-proBNP(1-108) monoclonal antibody (hinge 76
monoclonal antibody recognizing the epitope RAPR.sub.76S.sub.77P
(SEQ ID NO: 58), Bio-Rad) and assay buffer into a reaction vessel.
Then, after 11 minutes of incubation and wash cycles, the
anti-human BNP monoclonal antibody 20G7 conjugated to phycoerythrin
(PE) is added and incubated for 2 minutes. After removal of excess
conjugate, the bead mixture is passed through the detector which
identifies the dyed beads and the amount of antigens captured on
the beads by the fluorescence of PE. After calibration using a set
of six distinct calibrators, the three levels of quality controls
and patient samples results are expressed in pg/mL.
Two Quality Control beads are also tested with each sample to
enhance the integrity of the overall system.
14.3. Results:
The distributions of the BioPlex.TM. 2200 proBNP values for the
Control and the ischemic stroke populations are shown in Table 8
and FIG. 11. The level of proBNP(1-108) was significantly higher in
the ischemic stroke group compared to the control group
(Mann-Whitney, p<0.0001). The results demonstrate that the
proBNP(1-108) is also a useful plasma biomarker for the early
diagnosis of Ischemic stroke.
TABLE-US-00019 TABLE 8 BioPlex .TM. 2200 proBNP concentrations in
ischemic stroke and control citrated plasma samples (minimum,
1.sup.st quartile, median, 31.sup.rd quartile and maximum values).
Minimum 1.sup.st Quartile Median 3.sup.rd Quartile Maximum
Populations (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) Control
population 0 0 1 2 23 (N = 42) (IC95%: 0-2) Ischemic 2 34 71 219
1019 Stroke population (IC95%: 38-145) (N = 32)
The inventors clearly demonstrate that the proBNP(1-108) sandwich
assay using the monoclonal antibody 20G7 described in this
invention can measure proBNP(1-108) concentrations in patients with
stroke.
Example 15
proBNP(1-108) Assay in Patients Suffering from an Acute Coronary
Disorder
15.1. Samples: EDTA plasmas from 27 patients with an acute coronary
disorder (with Troponine I plasma mean values reaching 12.5.+-.6.9
ng/ml) originating from a commercial source (ProMedex, NY, USA)
EDTA plasmas from 48 healthy subjects (healthy volunteers,
ProMedex, NY, USA).
15.2. Materials and Methods
The materials and methods used for the BNP(1-32) assay were
identical to those described above in 8.2, and for the
proBNP(1-108) in 10.2.
15.3 Results
The proBNP(1-108) values obtained from plasmas of patients admitted
in emergency and diagnosed for an acute coronary disorder by means
of the proBNP(1-108) assay described above, were found to be
strongly correlated (r.sup.2=0.956) to those obtained with the
BNP(1-32) assay using the 20G7 antibody according to the invention
(FIG. 12). Levels of BNP(1-32) and proBNP(1-108) of patients with
an acute coronary disorder (668.+-.619 and 1,518.+-.1,533 pg/ml for
proBNP(1-108) and BNP(1-32) respectively and assayed according to
the invention) were higher than those of healthy subjects (37.+-.32
and 227.+-.172 pg/ml for proBNP(1-108) and BNP(1-32) respectively
and assayed according to the invention).
It is therefore very clear that the sandwich assay using the 20G7
antibody according to the invention (coupled to peroxidase) allows
proBNP(1-108) concentrations, which are proportional to the level
of BNP(1-32), to be measured. These results once again prove the
usefulness of assaying proBNP(1-108) and BNP(1-32) as markers, in
particular as markers of an acute coronary disorder.
Example 16
Assay of proBNP(1-108) in the Glycosylated Form Using the 20G7
Monoclonal Antibody
16.1. Materials: proBNP(1-108) (recombinant protein produced in E.
coli for the non-glycosylated form, and from reprogrammed HEK293
cells for the glycosylated form, HyTest, Finland) Maxisorp.TM.
96-well flat-bottomed microplate (Nunc, Denmark) PBS (phosphate
buffered saline) buffer, pH 7.4, Gibco tablets, ref: 18912-014
(Invitrogen) Tween.RTM.20 (Sigma-Aldrich, USA, #P1379) hinge 76
monoclonal antibody which recognises the hinge sequence of
proBNP(1-108): epitope RAPR.sub.76S.sub.77P (SEQ ID NO: 55)
(Giuliani et al., Clin. Chem., 52: 6, 1054-1061, 2006) 20G7
monoclonal antibody (Bio-Rad) coupled to peroxidase
16.2. Method:
16.2.1. Principle of the Analytical Assay of Glycosylated
proBNP:
A standard range of 20 to 10,000 pg/ml of glycosylated
proBNP(1-108) was prepared in a 0.1% PBS-T buffer. A standard range
of 20 to 10,000 pg/ml of non-glycosylated proBNP(1-108) was
prepared in the same way in a 0.1% PBS-T buffer.
The assay is based on the sandwich ELISA principle on a microplate,
using for capture on a solid phase the monoclonal antibody (hinge
76 antibody, from Bio-Rad), recognising the epitopic sequence
RAPR.sub.76S.sub.77P (SEQ ID NO: 55) of proBNP(1-108) and fixed by
passive adsorption by way of 100 .mu.l of 0.5 .mu.g/ml solution per
well.
100 .mu.l of a solution of the 20G7 monoclonal antibody according
to the invention coupled to peroxidase at a concentration of 0.5
.mu.g/ml, in solution in 0.1% PBS-T containing 1% milk
(semi-skimmed), were used as detection reagents. The rest of the
protocol was identical to that of the ELISA in Example 10. The
detection characteristics of the 20G7 antibody were thus compared
for the two forms of proBNP(1-108)--glycosylated and
non-glycosylated.
16.3. Results:
The results shown in Table 9 and FIG. 13 correspond to the
glycosylated proBNP(1-108) and non-glycosylated proBNP(1-108)
assays by means of immunoassay using the immobilised hinge 76
antibody and the 20G7 antibody for detection.
TABLE-US-00020 TABLE 9 optical density values at 490 nm from the
assay of each proBNP(1-108) form tested. proBNP non-glycosylated
glycosylated range (pg/ml) proBNP(1-108) proBNP(1-108) 10,000 4
3.833 5000 3.807 3.801 2,500 3.829 3.023 1,250 3.256 1.679 625
1.816 0.925 312.5 0.989 0.465 156 0.510 0.268 78 0.221 0.130 39
0.109 0.087 20 0.063 0.041 9 0.037 0.028 0 0.021 0.023
ProBNP(1-108) can be detected just as well in the non-glycosylated
form as in the glycosylated form by using 20G7. The
signal/background ratio is slightly greater for non-glycosylated
proBNP than for its glycosylated form (signal/background ratios of
3 and 1.8 respectively are obtained at 20 pg/ml).
Example 17
Immunoreactivity of proBNP(1-108) in its Glycosylated Form and
Implications for the proBNP(1-108) Assay with the Hinge 76
Antibody
17.1. Materials: ProteOn XPR36 analyser (surface plasmon resonance
SPR technology, Bio-Rad, USA) proBNP(1-108) (recombinant protein,
HyTest, Finland) in the glycosylated and non-glycosylated forms (13
to 200 nM) anti Fc fragment antibodies (Sigma, USA) monoclonal
hinge 76 antibody (Bio-Rad) which recognises the epitope
RAPR.sub.76S.sub.77P (SEQ ID NO: 55) and is used at a concentration
of 30 .mu.g/ml in 0.1% PBS-T PBS buffer (phosphate buffered
saline), pH 7.4
17.2. Method:
17.2.1. Principle:
The ProteOn XPR36 analyser (the principle of which is based on the
surface plasmon resonance technology (SPR)), was used to define the
kinetics and the affinity of the interaction of the 20G7 monoclonal
antibody and with glycosylated and non-glycosylated proBNP(1-108)
forms. The inventors followed the manufacturer's instructions. A
monoclonal antibody was immobilised on a biosensor (solid surface)
by using an anti Fc fragment antibody whilst the glycosylated or
non-glycosylated soluble antigen proBNP(1-108) circulated at
increasing concentrations (13 to 200 nM) in a constant flow on the
surface of the biosensor at room temperature. The angle at which
the SPR signal is detected is directly proportional to the
refractive index of the medium in which the evanescent wave
propagates. The variations in the refractive index are expressed in
resonance units (RU, where 1000 resonance units correspond to 1 ng
of fixed proteins per mm.sup.2 of surface area). The quantification
of the interactions and the affinity between the antigen and the
monoclonal antibody is assessed by calculating the association rate
constant (ka) and the dissociation rate constant (kd) by global
data processing using the device software (Bio-Rad). The
equilibrium dissociation constant (KD=kd/ka) in mol/l reflects the
affinity of the glycosylated or non-glycosylated proBNP(1-108)
antigen for the monoclonal antibody.
17.2.2. Results:
Table 10 shows the characteristics of the interaction between the
anti-hinge antibody (hinge 76 antibody with the epitope
RAPR.sub.76S.sub.77P (SEQ ID NO: 55) Bio-Rad) and glycosylated and
non-glycosylated proBNP(1-108). Although the affinity constant
between the anti-hinge antibody and non-glycosylated proBNP(1-108)
was greater (1.73.10.sup.-10) than that for glycosylated
proBNP(1-108) (2.35.10.sup.-8), the antibody targeting the hinge
region of proBNP(1-108) recognised both the glycosylated and
non-glycosylated forms with high affinity.
TABLE-US-00021 TABLE 10 Reactivity of the hinge 76 antibody towards
glycosylated and non- glycosylated proBNP(1-108) Hinge 76 antibody
ka kd KD = kd/ka (M.sup.-1s.sup.-1) (s.sup.-1) M non-glycosylated
proBNP(1-108) 1.12.10.sup.6 1.94.10.sup.-4 1.73.10.sup.-10
glycosylated proBNP(1-108) 1.17.10.sup.5 2.72.10.sup.-3
2.35.10.sup.-8
Example 18
Biepitopic and Triepitopic Calibrators
18.1. The structure of all of the biepitopic and triepitopic
calibrators according to the invention may be linear or branched,
provided that the immunoreactivity of the incorporated epitopes is
preserved.
The synthesis protocols which may be used to produce these
calibrators are those in the field of organic chemistry of peptides
well known to the one skilled in the art (in this context, see
"Peptide synthesis" in Example 1).
For the epitopes E.sub.2 and E.sub.3, the linear peptide sequences
according to the invention may be selected in a non-limiting manner
from the group consisting of the following sequences:
TABLE-US-00022 SEQ ID NO: 56: PRSPKMVQG SEQ ID NO: 57: APRSPKMV SEQ
ID NO: 58: SGLGCKLV SEQ ID NO: 59: SPKMVQGSG SEQ ID NO: 60:
YTLRAPRSPKMVG
18.2. Examples of Synthesised Epitopes According to the
Invention
The inventors synthesised the following calibrators:
18.2.1. Biepitopic Calibrators:
TABLE-US-00023 CaliproBNP1: (SEQ ID NO: 66 and SEQ ID NO: 64)
Ac-YTLRAPRSPKMV-Ahx-SFGRKMDRISS-CONH.sub.2 CaliproBNP2: (SEQ ID NO:
66 and SEQ ID NO: 65)
Ac-YTLRAPRSPKMV-Ahx-CFGRKMDRISSSSGLGCK-CONH.sub.2 CaliProBNP3: (SEQ
ID NO: 67 and SEQ ID NO: 51)
Ac-YTLRAPRSPKMVQG-Ahx-FGRKMDR-CONH.sub.2
These three biepitopic calibrators can be used to calibrate a
proBNP(1-108) assay, as described above in 10.2, based on the
immobilisation in a solid phase of the monoclonal hinge 76 antibody
that recognizes the RAPRSP (SEQ ID NO: 55) (Giuliani et al., supra)
and, in detection the monoclonal 20G7 antibody-peroxydase for
example.
TABLE-US-00024 (SEQ ID NO: 51 and SEQ ID NO: 68) CaliproBNP4:
Ac-FGRKMDR-Ahx-SGLGC*KVLRRH-COOH (SEQ ID NO: 51 and SEQ ID NO: 69)
CaliproBNP4b: Ac-FGRKMDR-Ahx-SGLGC*KVLR-CONH.sub.2
These two biepitopic calibrators can be used to calibrate a
BNP(1-32) assay, based on the immobilisation in a solid phase of
the monoclonal antibody directed the C-terminal portion of
BNP(1-32), such as for example monoclonal antibodies 50B7 or 50E1
(HyTest, Finland) and, in detection the monoclonal 20G7
antibody-peroxydase for example.
TABLE-US-00025 (SEQ ID NO: 59 and SEQ ID NO: 51) CaliproBNP5:
Ac-SPKMVQGSG-Ahx-FGRKMDR-CONH.sub.2
This biepitopic calibrator can be used to calibrate a BNP(1-32)
assay, as described above in 8.2, based on the immobilization in a
solid phase of the polyclonal antibody L21016 (Bio-Rad) and, in
detection the monoclonal 20G7 antibody-peroxydase for example. In
the calibrators sequences described above, the structural formula
of the Ahx group is NH--(CH.sub.2).sub.5--CO.
The binding group of formula --NH--(CH.sub.2).sub.5--CO-- is
derived from a well-known coupling agent, amino-hexanoic acid
(AHX), which enables to covalently couple two peptide sequences
together.
C*=C(Acm)=Cysteine blocked by an acetamidomethyl (protecting group,
well-known from the one skilled in the art).
18.2.2. Triepitopic Calibrators:
TABLE-US-00026 (SEQ ID NO: 66 and SEQ ID NO: 51 and SEQ ID NO: 68)
CaliproBNP6: Ac-YTLRAPRSPKMV-Ahx-FGRKMDR-Ahx-SGLGC*KVLRRH-COOH (SEQ
ID NO: 66 and SEQ ID NO: 51 and SEQ ID NO: 69) CAliproBNP6b:
Ac-YTLRAPRSPKMV-Ahx-FGRKMDR-Ahx-SGLGC*KVLR-CONH.sub.2
These two triepitopic calibrators can be used to calibrate both a
proBNP(1-108) and a BNP(1-32) assay, using antibodies such as the
monoclonal hinge 76 antibody that recognizes the sequence RAPRSP
(SEQ ID NO: 55) (Giuliani et al., supra) and the monoclonal 20G7
antibody of the invention and an antibody with an epitope directed
against the C-terminal portion of BNP(1-32) for example.
18.3. Materials and Methods
To depict the usefulness of these calibrators, results of
calibration and stabilities are displayed in two different formats,
BioPlex.TM. assay and ELISA assay. The proBNP(1-108) biepitopes,
the CaliproBNP1 and CaliproBNP3 compounds, were tested by means of
the BioPlex.TM. 2200 proBNP assay as described in Example 14. The
BNP(1-32) biepitope, the CaliproBNP5 compound, was tested by means
of the assay described in Example 8.2 and the triepitope, the
CaliproBNP6 compound was tested by means of the assay described in
Example 10.2.
18.4. Protocole
The different compounds were tested at different concentrations
diluted in 0.1 M succinate buffer, pH 7.6, containing 5% BSA, 2 mM
CaCl.sub.2, 10% antiproteases cocktail (Sigma reference P2714),
0.1% Proclin, 0.095% NaN.sub.3 and 0.1% sodium benzoate.
The CaliproBNP1 calibrator was diluted to 1 .mu.g/mL, 0.2 .mu.g/mL,
0.1 .mu.g/mL, and 0.02 .mu.g/mL, the CaliproBNP3 calibrator was
diluted to 1.6 .mu.g/mL, 0.8 .mu.g/mL, 0.3 .mu.g/mL, 0.06 .mu.g/mL,
the CaliproBNP5 and CaliproBNP6 calibrators were diluted to from
100 ng/ml to 0.01 ng/ml in the same 0.1 M succinate buffer, pH
7.6.
The compounds stability was studied in accelerated condition (room
temperature) compared to synthetic BNP(1-32) (Sigma-Aldrich,
Etats-Unis, #B-5900) and recombinant proBNP(1-108) (HyTest Ref.
8PRO8) in the following way:
Recombinant proBNP(1-108), synthetic BNP(1-32) and calibrator
peptides were diluted in 0.1 M succinate buffer, pH 7.6 described
above. Each solution was divided in 10 tubes placed at room
temperature (20.degree. C..+-.5.degree. C.). Then one tube of each
solution was frozen at J0, J+7, J+14, J+21. At J+21, the different
solutions were thawed and assayed by means of the BioPlex.TM. 2200
proBNP assay described in Example 14 or of the immunoassays
described in Examples 8.2 and 10.2.
In the ELISA assay, a range of proBNP(1-108), BNP(1-32) and
CaliproBNP6 from 25 ng/mL to 0.04 ng/mL was tested. For CaliBNP4
and CaliBNP5, the tested range was from 2 ng/mL to 0.004 ng/mL.
In the BioPlex.TM. 2200 proBNP assay, the stability of two
concentrations of proBNP(1-108), 10 ng/mL and 1 ng/mL, of
CaliproBNP1, 2 .mu.g/mL and 0.125 .mu.g/mL and CaliproBNP3, 1
.mu.g/mL and 0.3 .mu.g/mL, were analyzed. In ELISA format, three
concentrations of proBNP(1-108) and BNP(1-32), 1.56 ng/mL, 0.78
ng/ml and 0.39 ng/mL, and of CaliproBNP5, 62.5 pg/mL, 31 ng/mL and
15.6 ng/mL, were analysed.
18.5. Results
18.5.1 CaliproBNP1 and CaliproBNP3 Calibrators in the BioPlex.TM.
2200 proBNP Assay
The results of the assay of the CaliproBNP1 and CaliproBNP3
compounds in range of different concentrations are displayed in
FIGS. 14 and 15 respectively.
In a BioPlex.TM. 2200 proBNP assay, the CaliproBNP1 and CaliproBNP3
compounds enable to generate increasing signal with the compound
concentration. These compounds are therefore usable as calibrators
of the proBNP(1-108) assay, once standardized on the proBNP(1-108)
molecule.
The results of the accelerated stability test of the recombinant
proBNP(1-108) and the CaliproBNP1 and CaliproBNP3 compounds in
range of different concentrations are displayed in Table 11.
TABLE-US-00027 TABLE 11 Liquid stability* at room temperature** in
buffer*** Concentration expressed by the ratio "signal at J0 +
X/signal at J0" Compound (.mu.g/mL) J0 + 7 J0 + 14 J0 + 21
recombinant 0.001 0.79 0.65 0.51 proBNP(1-108) 0.01 0.85 0.72 0.69
CaliproBNP1 0.125 0.93 0.92 0.95 2 0.95 0.93 0.91 CaliproBNP3 0.3
0.91 0.98 1.15 1 0.92 1.02 1.06 *Norme of acceptance of
stabilities: to make the stability at day X after J0 acceptable,
the ratio signal at J0 + X/signal at J0 has to be equal to 1.00
.+-. 0.2. **Room temperature: 20.degree. C. .+-. 5.degree. C.
***0.1M succinate buffer, pH 7.6, containing 5% BSA, 2 mM
CaCl.sub.2, 10% antiproteases cocktail (Sigma reference P2714),
0.1% Proclin, 0.095% NaN3 and 0.1%. sodium benzoate.
Biepitopic calibrators CaliproBNP1 and CaliproBNP3 clearly show a
higher stability than recombinant proBNP(1-108).
18.5.2 CaliproBNP5 and CaliproBNP6 Calibrators in Immunoassays
Based on the Use of 20G7 Antibody
The results of the test of the CaliproBNP5 and CaliproBNP6
compounds in range of different concentrations are displayed in
FIGS. 16 and 17, respectively.
In the BNP(1-32) and the proBNP(1-108) assay, the biepitopic
compound CaliproBNP5 and the triepitopic compound CaliproBNP6
enable to generate a signal increasing with the compound
concentration. These compounds are therefore usable as calibrators
of the proBNP(1-108) and the BNP(1-32) assay.
The results of the accelerated stability assay of recombinant
proBNP(1-108), BNP(1-32) and the CaliproBNP5 compound, in range of
different concentrations are displayed in Table 12.
TABLE-US-00028 TABLE 12 Liquid stability* at room temperature** in
buffer*** Concentration expressed by the ratio "signal at J0 +
X/signal at J0" Compound (ng/mL) J0 + 7 J0 + 14 J0 + 21 recombinant
1.56 0.58 0.5 0.45 proBNP(1-108) 0.78 1.08 0.82 0.88 0.39 0.71 0.66
0.79 synthetic 1.56 0.71 0.7 0.55 BNP(1-32) 0.78 0.69 0.65 0.53
0.39 0.81 0.7 0.53 CaliproBNP5 0.0625 1.02 1.02 1.1 0.031 1.06 1.01
1.06 0.0156 1.1 1.01 1.10 *Norme of acceptance of stabilities: to
make the stability at day X after J0 acceptable, the ratio signal
at J0 + X/signal at J0 has to be equal to 1.00 .+-. 0.2. **Room
temperature: 20.degree. C. .+-. 5.degree. C. ***0.1M succinate
buffer, pH 7.6, containing 5% BSA, 2 mM CaCl.sub.2, 10%
antiproteases cocktail (Sigma reference P2714), 0.1% Proclin,
0.095% NaN3 and 0.1%. sodium benzoate.
Once again, the CaliproBNP5 biepitopic calibrator displays a higher
stability than recombinant proBNP(1-108) and BNP(1-32).
TABLE-US-00029 Summary table of the sequences SEQ ID NO: SEQUENCES
1 HPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQES
PRPTGVWKSREVATEGIRGHRKMVLYTLRAPRSPKMVQGSGCFG RKMDRISSSSGLGCKVLRRH 2
SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH 3
HPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQES
PRPTGVWKSREVATEGIRGHRKMVLYTLRAPR 4 TGCFGRKMDRISTSTAIGCKVL 5
SGCYGRKMDRISTSTAIGCKVL 6 SGCFGRKMDRISSSSGLGCKVL 7
SGCFGRKMDRIATSTAIGCKVL 8 FGRKMDR 9 GRKMDR 10 FGRKMD 11 RKMDRI 12
SPKMVQGSGCFGRKM 13 KMVQGSGCFGRKMDR 14 VQGSGCFGRKMDRIS 15
GSGCFGRKMDRISSS 16 GCFGRKMDRISSSSG 17 FGRKMDRISSSSGLG 18
RKMDRISSSSGLGCK 19 MDRISSSSGLGCKVL 20 RISSSSGLGCKVLRR 21 VQGSGCFGR
22 SPKMVQGSGC 23 MDRISSSSGLG 24 RKMDRI 25 RKMDRISS 26 SPKMVQGSGC 27
PKMVQGSGCF 28 KMVQGSGCFG 29 MVQGSGCFGR 30 VQGSGCFGRK 31 QGSGCFGRKM
32 GSGCFGRKMD 33 SGCFGRKMDR 34 GCFGRKMDRI 35 CFGRKMDRIS 36
FGRKMDRISS 37 GRKMDRISSS 38 RKMDRISSSS 39 KMDRISSSSG 40 MDRISSSSGL
41 DRISSSSGLG 42 RISSSSGLGC 43 ISSSSGLGCK 44 SSSSGLGCKV 45
SSSGLGCKVL 46 SSGLGCKVLR 47 SGLGCKVLRR 48 GLGCKVLRRH 49 GCFGRKM 50
CFGRKMD 51 FGRKMDR 52 GRKMDRI 53 RKMDRIS 54 SPKMV 55 RAPRSP 56
PRSPKMVQG 57 APRSPKMV 58 SGLGCKVL 59 SPKMVQGSG 60 YTLRAPRSPKMVG 61
FGRKMDRISSSS 62 AGRKMDR 63 GCFGRKMDRIS 64 SFGRKMDRISS 65
CFGRKMDRISSSSGLGCK 66 YTLRAPRSPKMV 67 YTLRAPRSPKMVQG 68 SGLGCKVLRRH
69 SGLGCKVLR
SEQUENCE LISTINGS
1
691108PRTHomo sapiens 1His Pro Leu Gly Ser Pro Gly Ser Ala Ser Asp
Leu Glu Thr Ser Gly1 5 10 15Leu Gln Glu Gln Arg Asn His Leu Gln Gly
Lys Leu Ser Glu Leu Gln 20 25 30Val Glu Gln Thr Ser Leu Glu Pro Leu
Gln Glu Ser Pro Arg Pro Thr 35 40 45Gly Val Trp Lys Ser Arg Glu Val
Ala Thr Glu Gly Ile Arg Gly His 50 55 60Arg Lys Met Val Leu Tyr Thr
Leu Arg Ala Pro Arg Ser Pro Lys Met65 70 75 80Val Gln Gly Ser Gly
Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser 85 90 95Ser Ser Gly Leu
Gly Cys Lys Val Leu Arg Arg His 100 105232PRTHomo sapiens 2Ser Pro
Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp1 5 10 15Arg
Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His 20 25
30376PRTHomo sapiens 3His Pro Leu Gly Ser Pro Gly Ser Ala Ser Asp
Leu Glu Thr Ser Gly1 5 10 15Leu Gln Glu Gln Arg Asn His Leu Gln Gly
Lys Leu Ser Glu Leu Gln 20 25 30Val Glu Gln Thr Ser Leu Glu Pro Leu
Gln Glu Ser Pro Arg Pro Thr 35 40 45Gly Val Trp Lys Ser Arg Glu Val
Ala Thr Glu Gly Ile Arg Gly His 50 55 60Arg Lys Met Val Leu Tyr Thr
Leu Arg Ala Pro Arg65 70 75422PRTArtificial sequenceproBNP derived
sequence 4Thr Gly Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Thr Ser
Thr Ala1 5 10 15Ile Gly Cys Lys Val Leu 20522PRTArtificial
sequenceproBNP derived sequence 5Ser Gly Cys Tyr Gly Arg Lys Met
Asp Arg Ile Ser Thr Ser Thr Ala1 5 10 15Ile Gly Cys Lys Val Leu
20622PRTArtificial sequenceproBNP derived sequence 6Ser Gly Cys Phe
Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly1 5 10 15Leu Gly Cys
Lys Val Leu 20722PRTArtificial sequenceproBNP derived sequence 7Ser
Gly Cys Phe Gly Arg Lys Met Asp Arg Ile Ala Thr Ser Thr Ala1 5 10
15Ile Gly Cys Lys Val Leu 2087PRTArtificial sequenceproBNP derived
sequence 8Phe Gly Arg Lys Met Asp Arg1 596PRTArtificial
sequenceproBNP derived sequence 9Gly Arg Lys Met Asp Arg1
5106PRTArtificial sequenceproBNP derived sequence 10Phe Gly Arg Lys
Met Asp1 5115PRTArtificial sequenceproBNP derived sequence 11Arg
Lys Met Asp Arg1 51215PRTArtificial sequenceproBNP derived sequence
12Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met1 5 10
151315PRTArtificial sequenceproBNP derived sequence 13Lys Met Val
Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp Arg1 5 10
151415PRTArtificial sequenceproBNP derived sequence 14Val Gln Gly
Ser Gly Cys Phe Gly Arg Lys Met Asp Arg Ile Ser1 5 10
151515PRTArtificial sequenceproBNP derived sequence 15Gly Ser Gly
Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser1 5 10
151615PRTArtificial sequenceproBNP derived sequence 16Gly Cys Phe
Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly1 5 10
151715PRTArtificial sequenceproBNP derived sequence 17Phe Gly Arg
Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly1 5 10
151815PRTArtificial sequenceproBNP derived sequence 18Arg Lys Met
Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys1 5 10
151915PRTArtificial sequenceproBNP derived sequence 19Met Asp Arg
Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu1 5 10
152015PRTArtificial sequenceproBNP derived sequence 20Arg Ile Ser
Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg1 5 10
15219PRTArtificial sequenceproBNP derived sequence 21Val Gln Gly
Ser Gly Cys Phe Gly Arg1 52210PRTArtificial sequenceproBNP derived
sequence 22Ser Pro Lys Met Val Gln Gly Ser Gly Cys1 5
102311PRTArtificial sequenceproBNP derived sequence 23Met Asp Arg
Ile Ser Ser Ser Ser Gly Leu Gly1 5 10246PRTArtificial
sequenceproBNP derived sequence 24Arg Lys Met Asp Arg Ile1
5258PRTArtificial sequenceproBNP derived sequence 25Arg Lys Met Asp
Arg Ile Ser Ser1 52610PRTArtificial sequenceproBNP derived sequence
26Ser Pro Lys Met Val Gln Gly Ser Gly Cys1 5 102710PRTArtificial
sequenceproBNP derived sequence 27Pro Lys Met Val Gln Gly Ser Gly
Cys Phe1 5 102810PRTArtificial sequenceproBNP derived sequence
28Lys Met Val Gln Gly Ser Gly Cys Phe Gly1 5 102910PRTArtificial
sequenceproBNP derived sequence 29Met Val Gln Gly Ser Gly Cys Phe
Gly Arg1 5 103010PRTArtificial sequenceproBNP derived sequence
30Val Gln Gly Ser Gly Cys Phe Gly Arg Lys1 5 103110PRTArtificial
sequenceproBNP derived sequence 31Gln Gly Ser Gly Cys Phe Gly Arg
Lys Met1 5 103210PRTArtificial sequenceproBNP derived sequence
32Gly Ser Gly Cys Phe Gly Arg Lys Met Asp1 5 103310PRTArtificial
sequenceproBNP derived sequence 33Ser Gly Cys Phe Gly Arg Lys Met
Asp Arg1 5 103410PRTArtificial sequenceproBNP derived sequence
34Gly Cys Phe Gly Arg Lys Met Asp Arg Ile1 5 103510PRTArtificial
sequenceproBNP derived sequence 35Cys Phe Gly Arg Lys Met Asp Arg
Ile Ser1 5 103610PRTArtificial sequenceproBNP derived sequence
36Phe Gly Arg Lys Met Asp Arg Ile Ser Ser1 5 103710PRTArtificial
sequenceproBNP derived sequence 37Gly Arg Lys Met Asp Arg Ile Ser
Ser Ser1 5 103810PRTArtificial sequenceproBNP derived sequence
38Arg Lys Met Asp Arg Ile Ser Ser Ser Ser1 5 103910PRTArtificial
sequenceproBNP derived sequence 39Lys Met Asp Arg Ile Ser Ser Ser
Ser Gly1 5 104010PRTArtificial sequenceproBNP derived sequence
40Met Asp Arg Ile Ser Ser Ser Ser Gly Leu1 5 104110PRTArtificial
sequenceproBNP derived sequence 41Asp Arg Ile Ser Ser Ser Ser Gly
Leu Gly1 5 104210PRTArtificial sequenceproBNP derived sequence
42Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys1 5 104310PRTArtificial
sequenceproBNP derived sequence 43Ile Ser Ser Ser Ser Gly Leu Gly
Cys Lys1 5 104410PRTArtificial sequenceproBNP derived sequence
44Ser Ser Ser Ser Gly Leu Gly Cys Lys Val1 5 104510PRTArtificial
sequenceproBNP derived sequence 45Ser Ser Ser Gly Leu Gly Cys Lys
Val Leu1 5 104610PRTArtificial sequenceproBNP derived sequence
46Ser Ser Gly Leu Gly Cys Lys Val Leu Arg1 5 104710PRTArtificial
sequenceproBNP derived sequence 47Ser Gly Leu Gly Cys Lys Val Leu
Arg Arg1 5 104810PRTArtificial sequenceproBNP derived sequence
48Gly Leu Gly Cys Lys Val Leu Arg Arg His1 5 10497PRTArtificial
sequenceproBNP derived sequence 49Gly Cys Phe Gly Arg Lys Met1
5507PRTArtificial sequenceproBNP derived sequence 50Cys Phe Gly Arg
Lys Met Asp1 5517PRTArtificial sequenceproBNP derived sequence
51Phe Gly Arg Lys Met Asp Arg1 5527PRTArtificial sequenceproBNP
derived sequence 52Gly Arg Lys Met Asp Arg Ile1 5537PRTArtificial
sequenceproBNP derived sequence 53Arg Lys Met Asp Arg Ile Ser1
5545PRTArtificial sequenceproBNP derived sequence 54Ser Pro Lys Met
Val1 5556PRTArtificial sequenceproBNP derived sequence 55Arg Ala
Pro Arg Ser Pro1 5569PRTArtificial sequenceproBNP derived sequence
56Pro Arg Ser Pro Lys Met Val Gln Gly1 5578PRTArtificial
sequenceproBNP derived sequence 57Ala Pro Arg Ser Pro Lys Met Val1
5588PRTArtificial sequenceproBNP derived sequence 58Ser Gly Leu Gly
Cys Lys Val Leu1 5599PRTArtificial sequenceproBNP derived sequence
59Ser Pro Lys Met Val Gln Gly Ser Gly1 56013PRTArtificial
sequenceproBNP derived sequence 60Tyr Thr Leu Arg Ala Pro Arg Ser
Pro Lys Met Val Gly1 5 106112PRTArtificial sequenceproBNP derived
sequence 61Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser1 5
10627PRTArtificial sequenceproBNP derived sequence 62Ala Gly Arg
Lys Met Asp Arg1 56311PRTArtificial sequenceproBNP derived sequence
63Gly Cys Phe Gly Arg Lys Met Asp Arg Ile Ser1 5
106411PRTArtificial sequenceproBNP derived sequence 64Ser Phe Gly
Arg Lys Met Asp Arg Ile Ser Ser1 5 106518PRTArtificial
sequenceproBNP derived sequence 65Cys Phe Gly Arg Lys Met Asp Arg
Ile Ser Ser Ser Ser Gly Leu Gly1 5 10 15Cys Lys6612PRTArtificial
sequenceproBNP derived sequence 66Tyr Thr Leu Arg Ala Pro Arg Ser
Pro Lys Met Val1 5 106714PRTArtificial sequenceproBNP derived
sequence 67Tyr Thr Leu Arg Ala Pro Arg Ser Pro Lys Met Val Gln Gly1
5 106811PRTArtificial sequenceproBNP derived sequence 68Ser Gly Leu
Gly Cys Lys Val Leu Arg Arg His1 5 10699PRTArtificial
sequenceproBNP derived sequence 69Ser Gly Leu Gly Cys Lys Val Leu
Arg1 5
* * * * *
References